Chimeric vaccine antigens for anaplasmosis

ABSTRACT

Provided herein are chimeric recombinant polypeptides (chimeritopes) for use in vaccines against Anaplasmosis, in assays for diagnosing Anaplasmosis and in assays for measuring antibody titers induced by vaccination. The chimeritopes comprise, for example, antigenic segments of three Anaplasma proteins (OmpA, AipA and Asp14) and a non-antigenic segment of a Borrelia Osp protein (e.g. OspC) that is 10 amino acids in length, proline rich and random coil in conformation. Compositions comprising the chimeritopes, optionally in combination with additional Anaplasma proteins of interest, are also provided, as are methods of using the compositions as vaccines and diagnostic tools.

SEQUENCE LISTING

This application includes as the Sequence Listing the complete contentsof the accompanying text file “Sequence.txt”, created Apr. 16, 2019,containing 65,536 bytes, hereby incorporated by reference.

BACKGROUND OF THE INVENTION Field of the Invention

The invention generally relates to recombinant chimeric polypeptidescomprising epitopes derived from Anaplasma antigens. In particular, theinvention provides i) recombinant chimeric epitope-based polypeptides(chimeritopes) comprising segments of three Anaplasma proteins (OmpA,AipA and Asp14) and a proline rich segment of a Borrelia protein orderivative thereof; and ii) compositions comprising the chimeritopes,optionally in combination with Anaplasma proteins P130 and APH_1235. Thecompositions are used as vaccines and diagnostic tools.

Description of Related Art

Anaplasma phagocytophilum (Aph) is a tick-transmitted, obligateintracellular bacterium of the family Anaplasmataceae. Several speciesof this family including A. marginale, A. platys, Ehrlichia chaffeensis,E. canis, and E. ruminatium can cause infections in humans, companionanimals, livestock and wild animals. Infections caused by this group ofpathogens are generally referred to as “anaplasmosis” or “ehrlichiosis”.In humans, the most serious form of anaplasmosis, referred to as humangranculocytic anaplasmosis (HGA), is caused by Aph. Anaplasmosis ischaracterized by fever leukopenia, thrombocytopenia, elevated serumtransaminase, and increased susceptibility to potentially fatalopportunistic infections. It is typically treated with doxycycline ortetracycline.

While antibiotics are generally effective for treatment, preventativestrategies that can block infection, such as vaccination are preferable.Vaccination has historically proven to be the most cost-effectiveapproach for the prevention of many infectious diseases. At the presenttime, there are no veterinary or human vaccines available for theprevention of Anaplasma and Ehrlichia infections. As the incidence oftick-borne disease continues to increase so is the demand forpreventative vaccines. Here we detail the development of unique vaccineantigens and vaccine formulations that can address the growing problemof anaplasmosis and related infections. The deployment of an effectivepreventative vaccine will significantly advance veterinary and humanhealth and alleviate the socioeconomic stress associated with tick-bornediseases.

The “gold standard” serologic test for diagnosis of HGA in humans is anindirect immunofluorescence assay (IFA). This assay must be performed atmultiple time points over a period of several weeks and can only beperformed in specialized reference laboratories. A limitation of the IFAassay is its specificity. The IFA is designed to assess increases inboth IgM and IgG. While IgG antibody responses can be very specific, IgMresponses are less so and have the potential to yield false-positivetest results. Enzyme immunoassay (EIA) are also used for diagnosis. EIAtests are not quantitative and provide a simple positive or negativeresult. In veterinary medicine, lateral flow-based point of care assaysare widely used for diagnosis of anaplasmosis and ehrlichiosis. It isclear that there is a pressing need for improved assays that are easierto conduct and that provide greater specificity and sensitivity.

SUMMARY OF THE INVENTION

Defined antigenic segments (i.e., epitopes) of three proteins producedby Anaplasma have been identified and demonstrated to play criticalroles in the adherence and invasion of mammalian cells by Anaplasma.Recombinant chimeric polypeptides comprised of these epitopes have beensuccessfully produced and demonstrated, upon vaccination, to elicitantibody responses that block Anaplasma entry into mammalian cells. Theunique vaccine antigens that have been developed are referred to as“chimeritopes”. Chimeritope stands for chimeric epitope-based proteins.The unique composition of chimeritope polypeptide/proteinsdifferentiates this class of novel proteins from simple chimericproteins. The term chimeric protein is most commonly used in referenceto fusion proteins that are comprised of several different full-lengthproteins, or extended segments thereof, that are joined together to forma single contiguous protein. The distinction between a “chimericprotein” and a “chimeritope” is important because they arecompositionally different. Chimeritopes are designed to only containsegments of a protein that are immunologically or functionally relevant(i.e., that elicit protective or neutralizing antibody responses).

Accordingly, the chimeritope vaccine antigens described herein arecomprised of epitopes derived from at least three specific Anaplasmaproteins: OmpA (Outer membrane protein A, AipA (Aph invasion protein A),and Asp14 (14-kDa Aph surface protein). The chimeritopes contain atleast one copy of epitopes, or segments thereof, derived from the OmpA,AipA and Asp14 proteins. In some aspects, the carboxy terminus of eachchimeritope includes a cap sequence having a random coil structure and ahigh proline content (e.g. 33% or greater) to protect the chimeritopefrom degradation. In additional aspects, the cap sequence is comprisedof e.g. a 10 amino acid domain derived from a Borrelia protein such asPVVAESPKKP (SEQ ID NO: 5), or a functional variant thereof e.g.PVVPPSPKKP (SEQ ID NO: 6) or PVVPPSPPKP_(SEQ ID NO: 7).

The chimeritopes are used as vaccine antigens to elicit protectiveantibody responses against Anaplasma (e.g. Aph) and other relatedbacteria. An advantage of chimeritopes is that they elicit antibodyresponses in vaccinated mammals to three independent targets that arepresented on the surface of Anaplasma bacteria. By delivering thechimeritopes in combination with Anaplasma P130 and APH_1235, thesynergistic effects of eliciting antibodies that target severaldifferent proteins are expanded. The chimeritopes are also used todetect antibody responses that develop during infection with Anaplasmaor to measure antibody titers after vaccination with the APchimeritopes.

Several different exemplary AP chimeritopes have been produced andtested for their immunogenicity and ability to block intracellularinvasion of host cells by Aph. As detailed below, the chimeritopes havebeen assigned simple designations (AP1, AP2, AP3, AP4, etc.) todifferentiate them. Specifically for the AP3 and AP4 chimeritopes, asecond version of these proteins was made (v2). The v1 and v2 variantsdiffer in that the order of a two amino acid motif is reversed in thesevariants. The designation v1 or v2 follows the AP # designation (i.e.,AP3v1, AP3v2 etc). The purpose of generating the v1 and v2 AP proteinswas to determine if minor changes in the amino acid sequence of one ofthe component epitopes (the OmpA epitope) influences functionalactivity.

The AP vaccine antigens provide protection through a unique mechanism.Antibodies that are produced as a result of vaccination orhyperimmmunization can bind to the surface of Anaplasma and block orattenuate it's ability adhere to and or enter mammalian cells. Thevaccination-induced antibodies thus inhibit the ability of theseobligate intracellular pathogens to establish an infection. A distinctand unique attribute of the AP chimeritopes, as opposed to commonsubunit single protein or protein chimeric based vaccines, is that theAP chimeritopes elicit antibody that binds to several different targetproteins on the bacterial cell surface. The impact of antibody bindingto multiple targets, as opposed to a single protein produced by thebacteria, is synergistic. Furthermore, by combining epitopes frommultiple proteins into one protein, the cost of production is reducedand quality control and formulation strategies simplified. Embodimentsof these recombinant AP chimeritope proteins delivered with or withoutadditional Aph proteins (P130 and APH_1235) include preventive vaccines,passive and active therapeutic vaccines, diagnostic antigens andantigens for measuring vaccine induced antibody levels in vaccinatedanimals.

It is an object of this invention to provide a recombinant, chimericpolypeptide comprising, at least one copy of an invasion domain/epitopeof Anaplasma OmpA, at least one copy of an invasion domain/epitope ofAnaplasma AipA, and at least one copy of an invasion domain/epitope ofAnaplasma Asp14. In some aspects, the invasion domain of Anaplasma OmpAhas a sequence GKYDLKGPGKKVILELEVQL (SEQ ID NO: 1) and/orGKYDLKGPGKKVILELVEQL (SEQ ID NO: 2). In other aspects, the invasiondomain of Anaplasma AipA has a sequence SLDPTQGSHTAENI (SEQ ID NO: 3).In additional aspects, the invasion domain of Anaplasma Asp14 has asequence LKLERAVYGANTPKES (SEQ ID NO: 4). In yet further aspects, therecombinant chimeric polypeptide or polypeptides further comprise atleast one copy of a cap sequence that is placed on the C-terminus of thechimeritopes to stabilize and protect against proteolytic degradation. Asuitable cap sequence is a high proline, random coil, non-immunogenicsequence such as the 10 amino acid segment derived from the BorelliaOspC protein. In some aspects, the C-terminal cap sequence motif isPVVAESPKKP (SEQ ID NO: 5). Other suitable cap sequences include but arenot limited to PVVPPSPKKP (SEQ ID NO: 6). and PVVPPSPPKP (SEQ ID NO: 7).

In some aspects, the amino acid sequence of the recombinant, chimericpolypeptide is selected from the group consisting of:

(SEQ ID NO: 8) GKYDLWGKYDLKGPGKKVILELEVQLSLDPTQGSHTAENILKLERAVYGANTPKESLKLERAVYGANTPKESSLDPTQGSHTAENIGKYDLKGPGKKVILELEVQLSLDPTQGSHTAENIGKYDLKGPGKKVILELEVQLLKLERAVYGANTPKESPVVAESPKKP; (SEQ ID NO: 9)GKYDLWGKYDLKGPGKKVILELVEQLSLDPTQGSHTAENILKLERAVYGANTPKESLKLERAVYGANTPKESSLDPTQGSHTAENIGKYDLKGPGKKVILELVEQLSLDPTQGSHTAENIGKYDLKGPGKKVILELVEQLLKLERAVYGANTPKESPVVAESPKKP; (SEQ ID NO: 10)LKLERWLKLERAVYGANTPKESGKYDLKGPGKKVILELEVQLSLDPTQGSHTAENIGKYDLKGPGKKVILELEVQLSLDPTQGSHTAENILKLERAVYGANTPKESLKLERAVYGANTPKESSLDPTQGSHTAENIGKYDLKGPGKKVILELEVQLPVVAESPKKP; (SEQ ID NO: 11)LKLERWLKLERAVYGANTPKESGKYDLKGPGKKVILELVEQLSLDPTQGSHTAENIGKYDLKGPGKKVILELVEQLSLDPTQGSHTAENILKLERAVYGANTPKESLKLERAVYGANTPKESSLDPTQGSHTAENIGKYDLKGPGKKVILELVEQLPVVAESPKKP; (SEQ ID NO: 12)GKYDLWGKYDLKGPGKKVILELEVQLSLDPTQGSHTAENILKLERAVYGANTPKESGKYDLKGPGKKVILELEVQLSLDPTQGSHTAENILKLERAVYGANTPKESGKYDLKGPGKKVILELEVQLSLDPTQGSHTAENILKLERAVYGANTPKESPVVAESPKKP; (SEQ ID NO: 13)GKYDLWGKYDLKGPGKKVILELVEQLSLDPTQGSHTAENILKLERAVYGANTPKESGKYDLKGPGKKVILELVEQLSLDPTQGSHTAENILKLERAVYGANTPKESGKYDLKGPGKKVILELVEQLSLDPTQGSHTAENILKLERAVYGANTPKESPVVAESPKKP; (SEQ ID NO: 14)GKYDLWGKYDLKGPGKKVILELEVQLGKYDLKGPGKKVILELEVQLGKYDLKGPGKKVILELEVQLSLDPTQGSHTAENISLDPTQGSHTAENISLDPTQGSHTAENILKLERAVYGANTPKESLKLERAVYGANTPKESLKLERAVYGANTPKESPVVAESPKKP; (SEQ ID NO: 15)GKYDLWGKYDLKGPGKKVILELVEQLGKYDLKGPGKKVILELVEQLGKYDLKGPGKKVILELVEQLSLDPTQGSHTAENISLDPTQGSHTAENISLDPTQGSHTAENILKLERAVYGANTPKESLKLERAVYGANTPKESLKLERAVYGANTPKESPVVAESPKKP; (SEQ ID NO: 16)GKYDLWGKYDLKGPGKKVILELEVQLSLDPTQGSHTAENILKLERAVYGANTPKESLKLERAVYGANTPKESSLDPTQGSHTAENIGKYDLKGPGKKVILELEVQLSLDPTQGSHTAENIGKYDLKGPGKKVILELEVQLLKLERAVYGANTPKES; (SEQ ID NO: 17)GKYDLWGKYDLKGPGKKVILELVEQLSLDPTQGSHTAENILKLERAVYGANTPKESLKLERAVYGANTPKESSLDPTQGSHTAENIGKYDLKGPGKKVILELVEQLSLDPTQGSHTAENIGKYDLKGPGKKVILELVEQLLKLERAVYGANTPKES; (SEQ ID NO: 18)LKLERWLKLERAVYGANTPKESGKYDLKGPGKKVILELEVQLSLDPTQGSHTAENIGKYDLKGPGKKVILELEVQLSLDPTQGSHTAENILKLERAVYGANTPKESLKLERAVYGANTPKESSLDPTQGSHTAENIGKYDLKGPGKKVILELEVQL; (SEQ ID NO: 19)LKLERWLKLERAVYGANTPKESGKYDLKGPGKKVILELVEQLSLDPTQGSHTAENIGKYDLKGPGKKVILELVEQLSLDPTQGSHTAENILKLERAVYGANTPKESLKLERAVYGANTPKESSLDPTQGSHTAENIGKYDLKGPGKKVILELVEQL; (SEQ ID NO: 20)GKYDLWGKYDLKGPGKKVILELEVQLSLDPTQGSHTAENILKLERAVYGANTPKESGKYDLKGPGKKVILELEVQLSLDPTQGSHTAENILKLERAVYGANTPKESGKYDLKGPGKKVILELEVQLSLDPTQGSHTAENILKLERAVYGANTPKES; (SEQ ID NO: 21)GKYDLWGKYDLKGPGKKVILELVEQLSLDPTQGSHTAENILKLERAVYGANTPKESGKYDLKGPGKKVILELVEQLSLDPTQGSHTAENILKLERAVYGANTPKESGKYDLKGPGKKVILELVEQLSLDPTQGSHTAENILKLERAVYGANTPKES; (SEQ ID NO: 22)GKYDLWGKYDLKGPGKKVILELEVQLGKYDLKGPGKKVILELEVQLGKYDLKGPGKKVILELEVQLSLDPTQGSHTAENISLDPTQGSHTAENISLDPTQGSHTAENILKLERAVYGANTPKESLKLERAVYGANTPKESLKLERAVYGANTPKES; and (SEQ ID NO: 23)GKYDLWGKYDLKGPGKKVILELVEQLGKYDLKGPGKKVILELVEQLGKYDLKGPGKKVILELVEQLSLDPTQGSHTAENISLDPTQGSHTAENISLDPTQGSHTAENILKLERAVYGANTPKESLKLERAVYGANTPKESLKLERAVYGANTPKES.In further aspects, the amino acid sequence of the recombinant,chimeric polypeptide is: (SEQ ID NO: 13)GKYDLWGKYDLKGPGKKVILELVEQLSLDPTQGSHTAENILKLERAVYGANTPKESGKYDLKGPGKKVILELVEQLSLDPTQGSHTAENILKLERAVYGANTPKESGKYDLKGPGKKVILELVEQLSLDPTQGSHTAENILKLERAVYGANTPKESPVVAESPKKP. or(SEQ ID NO: 15) GKYDLWGKYDLKGPGKKVILELVEQLGKYDLKGPGKKVILELVEQLGKYDLKGPGKKVILELVEQLSLDPTQGSHTAENISLDPTQGSHTAENISLDPTQGSHTAENILKLERAVYGANTPKESLKLERAVYGANTPKESLKLERAVYGANTPKESPVVAESPKKP.(SEQ ID NO: 25; APH_1235) MKGKSDSEIR TSSSIRTSSS DDSRSSDDST RIRASKTHPQAPSDNSSILS SEDIESVMRC LEEEYGQKLS SELKKSMREE ISTAVPELTRALIPLLASAS DSDSSSRKLQ EEWVKTFMAI MLPHMQKIVA STQG, and(SEQ ID NO: 24; P130)MFEHNIPDTY TGTTAEGSPG LAGGDFSLSS IDETRDETIE SHRGSSADDPGYISFRDQDG NVMSRFLDVY VANFSLRCKH SPYNNDRMET AAFSLTPDIIEPSALLQESH STQNNVEEAV QVTALECPPC NPVPAEEVAP QPSFLSRIIQAFLWLFTPSS TTDTAEDSKC NSSDTSKCTS ASSESLEQQQ ESVEVQPSVLMSTAPIATEP QNAVVNQVNT TAVQVESSII VPESQHTDVT VLEDTTETITVDGEYGHFSD IASGEHNNDL PAMLLDEADF TMLLANEESK TLESMPSDSLEDNVQELGTL PLQEGETVSE GNTRESLPTD VSQDSVGVST DLEAHSQEVETVSEVSTQDS LSTNISQDSV GVSTDLEAHS KGVEIVSEGG TQDSLSADFPINTVESESTD LEAHSQEVET VSEFTQDSLS TNISQDSVGV STDLEVHSQEVEIVSEGGTQ DSLSTNISQD SVGVSTDLEA HSQEVETVSE FTQDSLSTNISQDSVGVSTD LEVHSQEVEI VSEGGTQDSL STNISQDSVG VSTDLEAHSKGVEIVSEGGT QDSLSADFPI NTVESESTDL EAHSPEGEIV SEVSTQDAPSTGVE1RFMDR DSDDDVLAL.and/or a subfragment or segment thereof. In certain aspects, thesubfragment of SEQ ID NO: 24 is or includes residues 163 to 619.

Also provided are methods of eliciting an immune response to Anaplasmain a subject in need thereof, comprising administering to the subject anamount of the pharmaceutical composition as described herein that issufficient to elicit an immune response in the subject.

In some aspects, the immune response is a protective immune response.

Also provided are methods of blocking or attenuating the binding ofAnaplasma to mammalian cells in a subject in need thereof, comprisingadministering to the subject a pharmaceutical composition as describedherein, wherein the pharmaceutical composition is administered in anamount sufficient to elicit the production of antibodies that block orattenuate the binding of Anaplasma to mammalian cells in the subject.

Other features and advantages of the present invention will be set forthin the description of invention that follows, and in part will beapparent from the description or may be learned by practice of theinvention. The invention will be realized and attained by thecompositions and methods particularly pointed out in the writtendescription and claims hereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and B. Expression of AP3v2 in E. coli. Plasmids encoding theAP3v2 protein (SEQ ID NO: 12) were transformed into Escherichia colistrains and protein production induced. Aliquots of culture werecollected over time (lanes 2-5) and the cell lysates fractionated usinga 4-12% SDS-polyacrylamide gel. (A) shows the induction profile forsample ZRL309 (E. coli BL21(DE3)Star cells carrying the pET28b-AP3v2plasmid) and (B) shows the induction profile of sample ZRL311 (E. coliBL21 carrying the pFLEX30/AP3v2 plasmid). The proteins were visualizedby staining. The arrow indicates the migration position of the AP3v2chimeritope. Molecular weight (MW) standards are shown in lane 1. Theresults demonstrate that Ap3v2 can be readily expressed in E. coli usingdifferent plasmid expression vectors.

FIGS. 2A and B. Expression of AP4v2 in E. coli. Plasmids encoding theAP4v2 protein (SEQ ID NO: 14) were transformed into E. coli strains andprotein production induced. Aliquots of culture were removed over timeand fractionated using a 4-12% SDS-polyacrylamide gel. (A, lanes 2-4)and (B, lanes 2-6) show the protein profiles for samples ZRL310 (E. coliBL21(DE3)Star) cells carrying the pET28b-AP4v2 plasmid) and ZRL312 (E.coli BL21 carrying the pFLEX30/AP4v2 plasmid). Proteins were visualizedby staining. The arrow indicates the migration position of therecombinant AP4v2 chimeritope. Purified AP4v2 is shown in lane 6.Molecular weight (MW) standards are shown in lane 1. The resultsdemonstrate that Ap4v2 can be readily expressed in E. coli usingdifferent plasmid expression vectors. Further, the produced protein isstable and was readily purified to homogeneity.

FIG. 3. SDS-PAGE analysis of purified AP4v2. AP4v2 (SEQ ID NO: 14)derived from ZRL312 was purified and dialyzed into PBS. The protein waselectrophoresed on a 4-12% SDS-polyacrylamide gel. MW markers areindicated in lane 1 and the purified protein is shown in lane 2. Lane 3shows the purified protein after passage through a 0.2 um-sterilizationfilter. The arrow indicates the migration position of the recombinantAP4v2 chimeritope. The data demonstrate that Ap4v2 can be readilypurified and that the protein is amenable to the sterilizing filtrationsteps that are required in vaccine production.

FIG. 4. The amino acid sequences and basic properties of APH_1235 andP130 (APH_0032). The sequences of the P130 (SEQ ID NO: 24) and APH_1235(SEQ ID NO: 25) proteins were analyzed using ProtParam (see the websiteat web.expasy.org/protparam). The ProtParam analyses provide importantinformation about the general properties of proteins. P130 (alsoreferred to in the literature as APH_0032, GE130, or AmpB) contributesto Aph virulence and survival in host cells. APH_1235 is expressed bythe bacterium at high levels exclusively when it is in its infectious ordense core (DC) form. P130 and APH_1235 are important virulence factors.Based on the role that these proteins play in virulence and theiroverall properties, these proteins were produced and purified forinclusion in the vaccine formulation. Analyses detailing the enhancedprotection that results from co-delivering these proteins along with AP3and or AP4 as a vaccine formulation are detailed below.

FIG. 5A-D. Expression of APH_1235 and P130 in E. coli. Genes encodingthe APH_1235 (SEQ ID NO: 25) and P130 (SEQ ID NO: 24) proteins werecloned and protein production induced (A) and (B), respectively. Priorto induction, and 6 hrs post-induction, aliquots of each culture wereanalyzed by SDS-PAGE using ANYkDa precast gels (Biorad). Proteins werevisualized by staining. Cell lysates from pre- and post-induction areshown in lanes 1 and lane 2, respectively of (A) and (B). Thechimeritope proteins were then purified and reassessed by SDS-PAGE.Purified APH_1235 and P130 are shown in (C) and (D) respectively (Lane1=MW markers and Lane 2=purified protein). Arrows indicate the migrationpositions of the APH_1235 and P130 proteins in each figure. Bothproteins were successfully produced and purified allowing for theirassessment as vaccine candidates.

FIGS. 6A and B. Demonstration that the P130 and APH_1235 proteins areantigenic during natural infection in client owned canines. To determineif naturally infected client owned dogs develop antibody against theP130 (SEQ ID NO: 24) and APH_1235 (SEQ ID NO: 25) proteins, singledilution ELISA analyses were conducted. APH_1235 and two versions of theP130 protein were screened (A): i) full length P130 (SEQ ID NO: 24),referred to in FIG. 6A as “P130FL”; and ii) P130C, a subfragment of P130spanning a C-terminal portion of the protein (residues 163 to 619 of SEQID NO: 24). The reason for generating and testing the C-terminalfragment of P130 stems from the presence of high probability antigenicdeterminants in this region. The recombinant chimeritope polypeptides,AP3v1 (SEQ ID NO: 12) and AP4v1 (SEQ ID NO: 14) were also analyzed (B).The proteins were immobilized in the wells of 96 well ELISA plates usingstandard ELISA methods and screened with serum from healthy (−) orAph-infected (+) dogs. Absorbance was read using a plate reader at awavelength of 405 nM (A405). A405 values are shown for each figure. Notethat for the analyses of the AP3v1 and AP4v1 proteins, the Aph P44protein was included as a positive control for antibody binding. The APproteins were screened with serum from purpose-bred beagles that wereexperimentally infected with Aph. These analyses demonstrate that theP130 and APH_1235 are antigenic during a natural infection. Importantly,the results in (B) demonstrate that the domains/epitopes selected forinclusion in the AP proteins do elicit significant antibody responses aspresented by Aph cells. This finding provides further supportingevidence for their inclusion in the chimeritopes.

FIGS. 7A and B. Comparative analyses of the ability of antisera againstAP1v1, AP2v1, AP3v1 and AP4v1 to inhibit Aph infection of HL60 cells.Sera from dogs vaccinated with AP proteins were incubated at differentconcentrations (1:125; 1:25 and 1:5 final dilutions) with HL60 cells andAph cells. The purpose of this experiment was to determine if vaccineinduced antibody can block infection and do so in a dose-dependentmanner. After incubation, the percentage of HL60 cells that becameinfected (A) and the mean number of Aph vacuoles (ApVs) per cell (B) wasdetermined and the data graphed. Preimmune serum and antisera raisedagainst the Borrelia Osp (irrelevant antibody) served as negativecontrols (Bars 1 and 2, respectively). Bars 3, 4, 5 and 6 show theresults obtained with sera raised against AP1v1 (SEQ ID NO: 8), AP2v1(SEQ ID NO: 10), AP3v1 (SEQ ID NO: 12) and AP4v1 (SEQ ID NO: 14) at thedilutions indicated. Significance values relative to preimmune serum areindicated (*P<0.05; **P<0.01; ***P<0.001; ****P<0.0001; ns=notsignificant). The data reveal that all APv1 series proteins inhibit tovarying degrees the intracellular localization of Anaplasma.

FIGS. 8A and B. Comparative analysis of the inhibition of Aph infectionof HL60 cells by canine anti-AP3v1 and canine anti-AP4v1 antiseragenerated using different adjuvants. The influence of adjuvant on theability of antibody induced by vaccination with the APv1 series ofproteins to block Aph invasion and vacuoles number per cell wasassessed. The assays were conducted as detailed in FIGS. 7A and B. (A)indicates the percentage of infected cells and (B) indicates the meannumber of Aph vacuoles (ApVs) per cell. In each figure, bar graphdesignations are as follows: Bar 1—preimmune serum; Bar 2AP1v1+REHYDRAGEL®; Bar 3—AP1v1+QCT; Bar 4—AP2v1+REHYDRAGEL®; Bar5—AP2v1+QCT; Bar 6—AP3v1+REHYDRAGEL®; Bar 7—AP3v1+QCT; Bar 8—AP4v1+QCT;Bar 9—AP1v1, AP2v1, AP3v1, AP4v1+QCT. Statistically significant valuesrelative to preimmune serum are indicated (*P<0.05; **P<0.01;***P<0.001; ****P<0.0001; ns=not significant).

FIG. 9A-D. Antibody to P130 and APH_1235 enhance the blocking ability ofrat anti-AP4v1 antiserum. The blocking ability of antibody elicited inrats by vaccination with the antigens listed below was determined after24 h (A) and (B) or 72 h (C) and (D) post-infection. In (A) and (C) thedata are presented as “normalized percentage of infected cells”. In (B)and (D), the data are presented as the mean number of Aph vacuoles(ApVs) per 100 cells. In (A)-(D), bar graph designations are asfollows: 1) rat preimmune serum; 2) anti-Ap4v1(rat); 3) anti-P130(rabbit); 4) anti-APH_1235 (rabbit); 5) anti-Ap4v1+anti-P130; 6)anti-Ap4v1+anti-APH_1235; 7) anti-AP4v1+anti-P130+anti-APH_1235.Statistically significant (*P<0.05; **P<0.01; ***P<0.001; ****P<0.0001)values relative to preimmune serum are indicated; ns=not significant.Brackets designate whether or not the two samples that the bracketsdemarcate are statistically significantly different from each other.

FIG. 10A-D. Antibody to P130 and APH_1235 enhance the blocking abilityof canine anti-AP4v1 antiserum. The blocking ability of antibodyelicited in dogs by vaccination with the antigens listed below wasdetermined after 24 h (A) and (B) or 72 h (C) and (D) post-infection. In(A) and (C), the data are presented as “normalized percentage ofinfected cells”. In (B) and (D), the data are presented as the meannumber of Aph vacuoles (ApVs) per 100 cells. In (A)-(D), bardesignations are as follows: 1) canine preimmune serum; 2)anti-Ap4v1(canine); 3) anti-P130 (rabbit); 4) anti-APH_1235 (rabbit); 5)anti-Ap4v1+anti-P130; 6) anti-Ap4v1+anti-APH_1235; 7)anti-AP4v1+anti-P130+anti-APH_1235. Statistically significant(****P<0.0001) values relative to preimmune serum are indicated; ns=notsignificant. Brackets designate whether or not the two samples that thebrackets demarcate are statistically significant from each other.

FIGS. 11A and B. In vitro inhibition of Aph infection using ratanti-AP3v2 or anti-AP4v2 antisera. In vitro inhibition of Aph infectionby rat anti-AP3v2 (SEQ ID NO: 13) or AP4v2 (SEQ ID NO: 15) antiserum wasassessed. Aph organisms were incubated in the presence of rat anti-AP3v2or rat anti-AP4v2 antisera at varying dilutions. (A) presents theresults expressed as normalized % of infected cells and (B) indicatesthe mean number of Aph vacuoles (ApVs) per 100 cells. In each figure,bar designations are as follows: 1) rat preimmune; 2) rat anti-AP3v2; 3)rat anti-AP4v2. Statistically significant (**P<0.01; ***P<0.001;****P<0.0001) values relative to preimmune serum are indicated; ns=notsignificant.

FIGS. 12A and B. Blocking of Aph infection using different combinationsof anti-APv4, anti-P130, anti-APH_1235 and anti-P44 antisera. Aph wasincubated in the presence of 1:5 dilutions of the sera indicated below.In (A) and (B), the data are presented as the percentage of infectedcells and the mean number of ApVs per cell, respectively. In each of (A)and (B), the bar designations are as follows: 1) rat preimmune serum; 2)anti-AP4v2 (rat); 3) anti-APH_1235 (rabbit); 4) anti-P130 (rabbit); 5)anti-P44 (rabbit); 6) anti-AP4v2+anti-APH_1235; 7) anti-AP4v2+P130; 8)anti-AP4v2 AS+anti-P44; 9) anti-AP4v2+anti-APH_1235+anti-P130; 10)anti-AP4v2+anti-APH_1235+anti-P44; 11) anti-AP4v2+anti-P130+anti-P44.Statistically significant (**P<0.01; ****P<0.0001) values relative topreimmune serum are indicated; ns=not significant. Brackets designatewhether or not the two samples that the brackets demarcate arestatistically significantly different from each other. The datademonstrate that, in some aspects, an optimal antibody response in termsof both IgG titer and ability to block intracellular invasion isgenerated by vaccination with AP4v2, P130 and APH-1235 proteins.

DETAILED DESCRIPTION

The present disclosure provides novel anti-A naplasma vaccine antigensthat were developed using “chimeritope technology” i.e. they arechimeric epitope based recombinant polypeptides. The disclosure furtherprovides two Aph proteins (P130 and APH_1235) that when (optionally)delivered in combination with the novel chimeritopes enhance theprotective efficacy of the vaccine formulation. Vaccines which includethe chimeritopes are designed to block the ability of Anaplasma to bindto mammalian cells, and enter or invade those cells. Because Anaplasmais an obligate intracellular bacterium (i.e. it cannot survive freelyoutside of eukaryotic cells), lessening the ability of Anaplasma toinvade mammalian cells also leads to killing Anaplasma. As describedbelow in the Examples section, the vaccine antigens have beensuccessfully produced, and their immunogenicity has been demonstrated invivo. In addition, antibodies raised to these chimeric proteinsattenuate (e.g. decrease or lessen) Anaplasma adherence to and invasionof mammalian cells, and thus decrease the ability of Anaplasma bacteriato infect mammalian cells, and/or increase the ability to clear anexisting infection. In some aspects, the chimeritopes are used e.g. invaccine compositions that may or may not also include the APH P130 andAPH_1235 proteins, polypeptides or antigenic fragments thereof.

Definitions

“Anaplasma” as used herein refers to a genetically related group ofbacteria that includes A. phagocytophilum, A. marginale, A. platys, E.chaffeensis, E. canis, E. ruminatium, and other antigenically related orsimilar species.

Epitope: the part of a protein or antigen that is capable of elicitingan immune response (antibody production) and that is capable of bindingthe specific antibody produced by such a response. Epitopes are commonlyreferred to as the antigenic determinants of a protein.

Immunodominant epitope: The epitope on a molecule that induces adominant, or most intense, immune response. The immunodominant epitopemay elicit, for example, the greatest antibody titer during infection orimmunization, as measured by, for example, the fraction of reactivityattributable to a certain antigen or epitope in an enzyme-linkedimmunosorbant assay as compared with the total responsiveness to anantigen set or entire protein.

Chimeritope: custom designed recombinant polypeptides created in thelaboratory that are comprised of epitopes and/or specific proteinsegments derived from multiple different proteins or protein variants.In sharp contrast to natural antigenic proteins, chimeritopes can bedesigned to elicit antibodies that can target several different proteintargets and several different species of one or more genera of bacteriathat cause disease in mammals. For example, the chimeritopes describedherein elicit antibodies that target numerous proteins produced bynumerous species of Anaplasma that cause anaplasmosis in mammals, suchas humans, companion animals, wild canids, wildlife and others.Chimeritopes may be referred to herein as “recombinant, chimericpolypeptides”, “recombinant AP chimeritope proteins”, “recombinantchimeritope constructs”, etc.

Designed: The term “designed” as used herein refers to an amino acidsequence of a recombinant, chimeric polypeptide (“chimeritope”), or ofan individual epitope, that is altered as described herein, andtherefore is unlike a native amino acid sequence. The term “designed”refers to the property that such chimeritopes are man-made, syntheticand not from nature. Instead, they are non-naturally occurring and arethe result of an inventive procedure. Further, the phrase “not fromnature” means that the sequence is not present as a non-artificialsequence entry in a sequence database, for example in GenBank, EMBL-Bankor Swiss-Prot. These databases and other similar sequence databases arewell known to the person skilled in the art.

Invasion domain: An invasion domain is a region of a surface protein ofa pathogen that binds a host cell and mediates pathogen entry into thehost cell. In some cases, uptake of the pathogen results in theformation of a vacuole in which the intracellular pathogen will reside.The invasion domains of the disclosure are linear amino acid sequenceswithin Asp14, OmpA, or AipA that are found on the outer membrane of thebacteria Aph and other Anaplasmataceae family members, and can varyslightly from one family member to the next. Invasion domains may bereferred to herein as “epitopes”.

Linker sequences: short peptide sequences encoding functional units thatmay be engineered or otherwise added at the ends or within recombinantproteins, polypeptides, peptides of interest. Linker sequences may beused as “handles” for protein purification, as detectable signals ofexpression or binding to other proteins or macromolecules, to modulatetertiary structure, enhance immunogenicity or to protect againstproteolytic degradation of a recombinant protein. Examples of linkersequences include but are not limited to an amino acid spacer, an aminoacid linker, a signal sequence, a stop transfer sequence, atransmembrane domain, a domain of a protein that separates two epitopes,and a C- or N-terminal protein cap.

LINKER: a program to generate linker sequences for fusion proteins.Protein Engineering 13(5): 309-312, which is a reference that describesunstructured linkers. Structured (e.g. helical) sequence linkers mayalso be designed using, for example, existing sequences that are knownto have that secondary structure, or using basic known biochemicalprinciples to design the linkers.

Tags: Recombinant amino acid sequences that can be added to the N- orC-terminus of a recombinant protein for the purpose of identification orfor purifying the recombinant protein for subsequent uses. Examples ofrecombinant protein tags that may be useful in practicing the inventioninclude but are not limited to glutathione-S-transferease (GST),poly-histidine, maltose binding protein (MBP), FLAG, V5, halo, myc,hemaglutinin (HA), S-tag, calmodulin, tag, streptavidin binding protein(SBP), SOFTAG1™, SOFTAG3™, Xpress tag, isopeptag, Spy Tag, biotincarboxyl carrier protein (BCCP), GFP, Nus-tag, strep-tag, thioredoxintag, TC tag, and Ty tag. All such tags are well-known to those ofordinary skill in the art of recombinant protein production.

Chimeric or fusion peptide/polypeptide: a recombinant or syntheticpeptide or polypeptide whose primary sequence comprises two or morelinear amino acid sequences which do not occur together in a singlemolecule in nature. The two or more sequences may, for example, encodefusions of full-length proteins or fusions of extended polypeptides, ortwo or more peptides (which may be the same or different) which areeither contiguous or separated by a linker sequences, etc.

Tandem repeats: two or more copies of nucleic acid or amino acidsequences encoding the same peptide, which are arranged in a linearmolecule and are either contiguous or separated by a linker sequences,etc.

Original, native or wild-type sequence: The sequence of a peptide,polypeptide, protein or nucleic acid as found in nature.

Recombinant peptide, polypeptide, protein or nucleic acid: peptide,polypeptide, protein or nucleic acid that has been removed from itsnative source (or is a copy of a sequence from a native source) andproduced and/or manipulated using molecular biology/genetic engineeringtechniques such as cloning, polymerase chain reaction (PCR), etc.

Synthetic peptide, polypeptide, protein or nucleic acid: peptide,polypeptide, protein or nucleic acid that has been produced usingchemical synthesis procedures.

The Constructs

The Anaplasma chimeritope constructs disclosed herein comprise antigenicsegments, or variants thereof, of at least three proteins: Anaplasmaproteins OmpA, AipA and Asp14. In addition, in some aspects, theproteins possess a cap sequence (e.g. a 10 amino acid cap sequence) attheir C-terminus. The cap sequence may be derived from e.g. a Borreliaouter surface protein or another suitable protein, or may be entirelysynthetically designed with no natural counterpart. Exemplary segmentsand/or variants thereof that are present in the chimeritopes are listedin Table 1 below, together with an indication of the origin, an assignednumber or letter designation and the associated SEQ ID NO.

TABLE 1 SEQ Designa- ID Origin tion Sequence NO: Anaplasma OmpA #1 OmpAGKYDLKGPGKKVILELEVQL 1 GKYDLKGPGKKVILELVEQL 2 Anaplasma AipA #2 AipASLDPTQGSHTAENI 3 Anaplasma Asp14 #3 Asp14 LKLERAVYGANTPKES 4Borrelia Osp #C-C10 PVVAESPKKP 5 Exemplary variant PVVPPSPKKP 6of SEQ ID NO: 5 Exemplary variant PVVPPSPPKP 7 of SEQ ID NO: 5

In some aspects, the recombinant chimeritope construct has a single copyof each antigenic segment joined together in a polypeptide. However, tofacilitate production and/or to increase antigenicity, generallymultiple copies of each Anaplasma segment are present. Thus, multiplecopies of one or more of each segment may be present, e.g. from about 1to about 20 copies of each, such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 copies. In some aspects, therecombinant polypeptides encompassed herein comprise from e.g. at leastabout 1 to about 5 or more copies (e.g. about 1, 2, 3, 4, or 5 or morecopies) of the #1 OmpA, #2 AipA and #3 Asp14 epitopes.

The number of copies of each Anaplasma based segment that is present mayor may not be the same for all segments. For example, two copies of eachof #1 OmpA and #2 AipA may be present in a recombinant construct thathas 3 or 4 copies of #3 Asp14; or one copy of #3 Asp14 may be present ina construct that comprises 2 copies of #1 OmpA and 4 copies of #2 AipA,and so on. All such constructs are encompassed herein. In some aspects.3 copies of each of #1 OmpA, #2 AipA and #3 Asp14 are present in aconstruct. Generally only one copy of #C-C10 (or a variant thereof) ispresent in each protein. The function of the C10 segment is to provide aprotective cap at the C-terminus that is non-immunogenic, and thatinhibits proteolytic degradation of the chimeritope proteins.

The Anaplasma epitopes may be in any linear order in a chimeritope, i.e.the position of one or more epitopes and/or other elements within aconstruct may be “swapped” or “exchanged”, compared to the exemplaryproteins disclosed herein. For example, the order of the one or morecopies of the segments may be, when reading from the segment nearest tothe amino terminus of the protein toward the carboxyl terminus: #1 OmpA,#2 AipA, #3 Asp14; or #2 AipA, #1 OmpA, #3 Asp1; or #3 Asp1, #2 AipA, #1OmpA; and so on. Further, if multiple copies of a segment are present,they may be present in tandem, e.g. #1 OmpA, #1 OmpA, #1 OmpA; #2 AipA,#2 AipA, #2 AipA; #3 Asp14, #3 Asp14, #3 Asp14; etc.; or they may not bein tandem, e.g. they may be interspersed within other segments, e.g. #1OmpA, #2 AipA, #3 Asp14; #10 mpA, #2 AipA, #3 Asp14; #10 mpA, #2 AipA,#3 Asp14; etc.

The amino acid sequences of the antigenic segments and the exemplarychimeritopes disclosed herein may be altered and still be suitable foruse. In other words, the sequences need not be identical to thesequences as disclosed herein by SEQ ID NO. For example, certainconservative amino acid substitutions are made without having adeleterious effect on the ability of an individual epitope or achimeritope as a whole to elicit an immune response, e.g. a protectiveimmune response. A conservative substitution is one in which an aminoacid is substituted for another amino acid that has similar properties,such that one skilled in the art of peptide chemistry would expect thesecondary structure and hydropathic nature of the polypeptide to besubstantially unchanged. In some exemplary aspects, the following groupsof amino acids represent conservative exchanges/substitutions: aliphatic(glycine, alanine, valine, leucine, isoleucine); hydroxyl orsulfur/selenium-containing (serine, cysteine, selenocysteine, threonine,methionine); aromatic (e.g. phenylalanine, tyrosine, tryptophan); basic(histidine, lysine, arginine); and acidic (aspartate, glutamate) andtheir amides (asparagine glutamine) For example, conservativesubstitutions such as the following may be tolerated: substitution ofone positively charged amino acid for another positively charged aminoacid; substitution of a negatively charged amino acid for anothernegatively charged amino acid; substitution of a hydrophobic amino acidfor another hydrophobic amino acid; etc. In fact, the results presentedherein have demonstrated that other non-conservative minor alterationsof amino acid sequence (e.g. the reversal of the sequence EV to VE) donot inhibit or alter the ability of the AP proteins to elicit Ab thatcan block infection. Specifically, this was demonstrated by comparingimmune responses of AP3v1 with AP3v2 and AP4v1 with AP4v2 (see Table 1).All such substitutions, alterations or variants are encompassed herein,as long as the resulting sequence still functions to elicit a suitableimmune response, and/or to detect antibodies in biological samples, asdescribed herein.

Versions of the sequences presented herein with one or more deletionsare also encompassed, e.g. versions from which about 1-5 (e.g. about 1,2, 3, 4, or 5) consecutive amino acids have been deleted, are alsoencompassed, as long as the physiological function of the individualepitope, or the full length chimeritope (e.g. the ability to elicit animmune response and/or detect antibodies in biological samples) is notimpaired. Such deletions may be truncations e.g. located at the amino orcarboxyl terminus, or internal deletions within a sequence.

In addition, in some aspects, altered or variant sequences may containan insertion of e.g. from about 1-5 amino acids (e.g. 1, 2, 3, 4, or 5amino acids), and still be tolerated, as long as the physiologicalfunction of the individual epitope, or the full length chimeritope (e.g.the ability to elicit an immune response), is not impaired. Insertionsmay be made e.g. at the amino terminus, the carboxyl terminus, within asequence, or between epitope sequences.

Amino acid sequences that are substituted, truncated or have aninsertion are typically referred to herein as “based on” or “derivedfrom” or “variants of” the original sequence.

Examples of changes/variations include but are not limited to:elimination or introduction of a protease cleavage site; elimination orintroduction of a lipidation sequence; changes which increase ordecrease solubility (e.g. changes to hydrophobicity, etc.); changeswhich increase or decrease intra- or inter-molecular interactions suchas folding, ionic interactions, salt bridges, the formation of disulfidebonds, the formation of multimers (e.g. dimers, trimers, etc.); and soon, which are effected by adding or removing one or more amino acidsthat participate in such interactions. In some aspect, the changes avoidor decrease such interactions; in other aspects, the changes promote orincrease such interactions. For example, the introduction of one or morecysteine residues can permit the formation of disulfide bridges within asequence, thereby stabilizing the sequence, e.g. in vivo. Similarly, theintroduction of one or more lipidation sequences may confer desirableproperties such as optimal folding, antigenicity, solubility, etc.Changes may be introduced which prevent interference with thepresentation and accessibility of the individual epitopes along thelength of the chimera, or which increase such accessibility, e.g.placement of a sequence at the surface of a folded construct. All suchchanges are intended to be encompassed by the present invention, so longas the resulting amino acid sequence functions to elicit an immuneresponse, e.g. a protective immune response, in at least one targetedmammalian population.

In general, altered (variant) sequences exhibit at least about 50% to99% identity or similarity to a corresponding sequence in the nativeprotein, e.g. about 60 to 70, or 70 to 80, or 80 to 90, or 90 to 99%identity/similarity (e.g. about 90, 91, 92, 93, 94, 95, 96, 98, or 99%)to the wild type sequence. “Identity” defines the percentage of aminoacids with a direct match in a sequence alignment; percent similarity oftwo sequences is the sum of both identical and similar matches (residuesthat have similar properties). In other words, percent identity refersto the percentage of identical residues while percent similarity refersto the percentage of residues with similar physicochemical properties.In some aspects, the altered sequence is about 95 to 100% identical orsimilar, e.g. about 95, 96, 97, 98 or 99% identical/similar. Variantpolypeptides may have one or more conservative amino acid variations orother minor modifications and retain biological activity, i.e., arebiologically functional equivalents. A biologically active equivalenthas substantially equivalent function when compared to the correspondingoriginal polypeptide. For example, as shown herein AP3v1 and Ap3v2 aswell as AP4v1 and AP4v2 are biologically functional equivalents (e.g.,replacing the EV motif with VE did not affect immunological properties).

Percent sequence identity or similarity has an art recognized meaningand there are a number of methods to measure identity/similarity betweentwo polypeptide or polynucleotide sequences. See, e.g., Lesk, Ed.,Computational Molecular Biology, Oxford University Press, New York,(1988); Smith, Ed., Biocomputing: Informatics And Genome Projects,Academic Press, New York, (1993); Griffin & Griffin, Eds., ComputerAnalysis Of Sequence Data, Part I, Humana Press, New Jersey, (1994); vonHeinje, Sequence Analysis In Molecular Biology, Academic Press, (1987);and Gribskov & Devereux, Eds., Sequence Analysis Primer, M StocktonPress, New York, (1991). Methods for aligning polynucleotides orpolypeptides are codified in computer programs, including the GCGprogram package (Devereux et al., Nuc. Acids Res. 12:387 (1984)),BLASTP, BLASTN, FASTA (Atschul et al., J. Molec. Biol. 215:403 (1990)),and Bestfit program (Wisconsin Sequence Analysis Package, Version 8 forUnix, Genetics Computer Group, University Research Park, 575 ScienceDrive, Madison, Wis. 53711) which uses the local homology algorithm ofSmith and Waterman (Adv. App. Math., 2:482-489 (1981)). For example, thecomputer program ALIGN which employs the FASTA algorithm can be used.

Variant polypeptides can generally be identified by modifying one of thepolypeptide sequences of the disclosure, and evaluating the propertiesof the modified polypeptide to determine if it is a biologicalequivalent. A variant is a biological equivalent if it retains e.g. 90%or greater, of the activity of the original polypeptide (e.g. retainsthe ability to elicit an immune response and/or bind to Anaplasmaantibodies), as measured e.g. in a competition assay wherein thebiologically equivalent polypeptide is capable of reducing binding ofthe polypeptide of the disclosure to a corresponding reactive antigen orantibody by about 80, 85, 90, 95, 99, or 100%.

In some aspects, the individual epitopes in the chimeritopes areseparated from one another by one or more intervening sequences that arenot associated with an epitope disclosed herein in nature and aresubstantially neutral in character and, i.e. they do not necessarily inand of themselves elicit an immune response. Such sequences may or maynot be present between the epitopes. An amino acid spacer can comprisee.g. about 1, 5, 10, 20, 100, or 1,000 amino acids. If present, theymay, for example, separate the epitopes and contribute to stericisolation of the epitopes from each other. Alternatively, such sequencesmay be simply artifacts of recombinant processing procedures, e.g.cloning procedures. Such sequences are typically known as linker orspacer peptides (elements, sequences), many examples of which are knownto those of skill in the art. Suitable peptide linker sequences may bechosen, for example, based on the following factors: 1) the ability toadopt a flexible extended conformation; 2) the resistance to adopt asecondary structure that could interact with epitopes; and 3) the lackof hydrophobic or charged residues that might react with the epitopes.For example, peptide linker sequences may contain Gly, Asn and Serresidues. Other near neutral amino acids, such as Thr and Ala may alsobe used in the linker sequence. Amino acid sequences which may beusefully employed as linkers include those disclosed in, for example,Maratea et al., Gene, 1985, 40, 39-46; Murphy et al., Proc. Natl. Acad.Sci. USA, 1986, 83, 8258-8262; and U.S. Pat. No. 4,935,233, the completecontents of which is herein incorporated by reference in entirety;Crasto, C. J. and J. A. Feng. 2000; LINKER: a program to generate linkersequences for fusion proteins; Protein Engineering 13(5): 309-312, whichis a reference that describes unstructured linkers. Structured (e.g.helical) sequence linkers may also be designed using, for example,existing sequences that are known to have that secondary structure, orusing basic known biochemical principles to design the linkers.

Other elements may be present in the chimeritopes, for example signal orleader sequences that co-translationally or post-translationally directtransfer of the protein and/or sequences that “tag” the protein tofacilitate purification or detection of the protein. Examples of suchelements include but are not limited to: tryptophan residues, histidinetags, glutathione-S-transferase, trpE, maltose binding protein,Staphylococcal protein A, detection tags (e.g. S-tag, or Flag-tag),other antigenic amino acid sequences such as known Tv2ell epitopecontaining sequences, protein stabilizing motifs, sequences that enhancebinding of the polypeptide to a solid support (e.g. an immunoglobulin Fcregion or bovine serum albumin), etc. Amino terminus protecting groupssuch as acetyl, propyl, succinyl, benzyl, benzyloxycarbonyl ort-butyloxycarbonyl may be present, as may carboxyl terminus protectinggroups such as amide, methylamide, and ethylamide. In addition, thechimeric proteins may be chemically modified, e.g. by amidation,sulfonylation, lipidation, or other techniques that are known to thoseof skill in the art. Polypeptide stability can be enhanced by adding,for example, polyethylene glycol to the amino or carboxyl terminus ofthe polypeptide.

In another iteration, a bacterial lipidation motif or isolated Cysresidue could be added to the N-terminus of the protein to allow for itslipidation. The attachment of a lipid group can in some cases triggerstronger antibody responses.

An amino acid sequence as disclosed herein can also be linked to amoiety (i.e., a functional group that is a polypeptide or othercompound) that enhances an immune response (e.g., cytokines such asIL-2).

A chimeritope may also be designed to contain W (tryptophan) residueswith or without additional accompanying amino acid residues that are notnaturally found in the epitopes used to make the protein. The purpose ofincluding the W residue(s) is to make the protein detectable by UV andthus make quantitation of the protein easier and more accurate.Generally, such a W residue is introduced near the N-terminus of aconstruct but could also be introduced at the juncture of individualepitopes within the chimeritopes constructs. Examples of suitable short,W containing sequences include, but are not limited to: LKLERW (SEQ IDNO: 6) and GKYDLW (SEQ ID NO: 7). Note that the context in which the Wis introduced (i.e., alone or with one or more amino acid residues) doesnot need to be strictly defined as any sequence including a W could beused and it can vary in length.

A chimeritope can also have an amino acid or chemical moiety attached atone or both of its termini (N- and C-terminus) that functions tostabilize the protein and to protect the protein from proteolyticdegradation. We refer to such a protective sequence or moiety as a“cap”. Generally, cap sequences are about 10 amino acids in length (e.g.from about 5 to about 15 amino acids, such as about 5, 6, 7, 8, 9, 10,11, 12, 13, 14, or 15 amino acids). A typical cap sequence is rich inproline (e.g. is about 25 to 40% proline, such as about 25, 30, 35, or40% proline, such as about 33% proline) and adopts a random coilconfirmation. Also, cap sequences are typically not immunogenic. In someaspects, the constructs include the cap sequence PVVAESPKKP (SEQ ID NO:5). This sequence is derived from the last ten amino acid residues of aBorrelia Osp protein. It is added to the AP proteins to provide aC-terminal cap to protect against proteolytic degradation. This sequenceis particularly useful for this purpose because it is not immunogenicand thus does not elicit irrelevant antibody responses. Variants of thissequence are also encompassed, e.g. variants such as PVVPPSPKKP (SEQ IDNO: 6), and PVVPPSPPKP (SEQ ID NO: 7).

Exemplary Constructs

The sequences shown below represent examples of the recombinantchimeritopes disclosed herein. It is noted that the difference betweenthe “v1” AP chimeritope constructs and the “v2” AP chimeritopesconstructs is that the v1 constructs contain the EV sequence at theunderlined positions of the Omp epitope while the v2 constructs containthe sequence VE at those positions. Sequences containing a W andsequences from a Borrelia Osp are shown in bold.

AP1v1 construct: 1-2-3-3-2-1-2-1-3-C (note that the numbers listedindicate the specific epitopes and their order in each construct; thenumbering used is detailed in Table 1 above)

(SEQ ID NO: 8) GKYDLWGKYDLKGPGKKVILELEVQLSLDPTQGSHTAENILKLERAVYGANTPKESLKLERAVYGANTPKESSLDPTQGSHTAENIGKYDLKGPGKKVILELEVQLSLDPTQGSHTAENIGKYDLKGPGKKVILELEVQLLKLERAVYGANTPKESPVVAESPKKP.AP1v2 construct: 1-2-3-3-2-1-2-1-3-C (SEQ ID NO: 9)GKYDLWGKYDLKGPGKKVILELVEQLSLDPTQGSHTAENILKLERAVYGANTPKESLKLERAVYGANTPKESSLDPTQGSHTAENIGKYDLKGPGKKVILELVEQLSLDPTQGSHTAENIGKYDLKGPGKKVILELVEQLLKLERAVYGANTPKESPVVAESPKKP.AP2v1 construct: 3-1-2-1-2-3-3-2-1-C (SEQ ID NO: 10)LKLERWLKLERAVYGANTPKESGKYDLKGPGKKVILELEVQLSLDPTQGSHTAENIGKYDLKGPGKKVILELEVQLSLDPTQGSHTAENILKLERAVYGANTPKESLKLERAVYGANTPKESSLDPTQGSHTAENIGKYDLKGPGKKVILELEVQLPVVAESPKKP.AP2v2 construct: 3-1-2-1-2-3-3-2-1-C (SEQ ID NO: 11)LKLERWLKLERAVYGANTPKESGKYDLKGPGKKVILELVEQLSLDPTQGSHTAENIGKYDLKGPGKKVILELVEQLSLDPTQGSHTAENILKLERAVYGANTPKESLKLERAVYGANTPKESSLDPTQGSHTAENIGKYDLKGPGKKVILELVEQLPVVAESPKKP.AP3v1 construct: 1-2-3-1-2-3-1-2-3-C (SEQ ID NO: 12)GKYDLWGKYDLKGPGKKVILELEVQLSLDPTQGSHTAENILKLERAVYGANTPKESGKYDLKGPGKKVILELEVQLSLDPTQGSHTAENILKLERAVYGANTPKESGKYDLKGPGKKVILELEVQLSLDPTQGSHTAENILKLERAVYGANTPKESPVVAESPKKP.AP3v2 construct: 1-2-3-1-2-3-1-2-3-C (SEQ ID NO: 13)GKYDLWGKYDLKGPGKKVILELVEQLSLDPTQGSHTAENILKLERAVYGANTPKESGKYDLKGPGKKVILELVEQLSLDPTQGSHTAENILKLERAVYGANTPKESGKYDLKGPGKKVILELVEQLSLDPTQGSHTAENILKLERAVYGANTPKESPVVAESPKKP.AP4v1 construct: 1-1-1-2-2-2-3-3-3-C (SEQ ID NO: 14)GKYDLWGKYDLKGPGKKVILELEVQLGKYDLKGPGKKVILELEVQLGKYDLKGPGKKVILELEVQLSLDPTQGSHTAENISLDPTQGSHTAENISLDPTQGSHTAENILKLERAVYGANTPKESLKLERAVYGANTPKESLKLERAVYGANTPKESPVVAESPKKP.AP4v2 construct: 1-1-1-2-2-2-3-3-3-C (SEQ ID NO: 15)GKYDLWGKYDLKGPGKKVILELVEQLGKYDLKGPGKKVILELVEQLGKYDLKGPGKKVILELVEQLSLDPTQGSHTAENISLDPTQGSHTAENISLDPTQGSHTAENILKLERAVYGANTPKESLKLERAVYGANTPKESLKLERAVYGANTPKESPVVAESPKKP.AP5v1 construct: 1-2-3-3-2-1-2-1-3 (SEQ ID NO: 16)GKYDLWGKYDLKGPGKKVILELEVQLSLDPTQGSHTAENILKLERAVYGANTPIKESLKLERAVYGANTPKESSLDPTQGSHTAENIGKYDLKGPGKKVILELEVQLSLDPTQGSHTAENIGKYDLKGPGKKVILELEVQLLKLERAVYGANTPKES.AP5v2 construct: 1-2-3-3-2-1-2-1-3 (SEQ ID NO: 17)GKYDLWGKYDLKGPGKKVILELVEQLSLDPTQGSHTAENILKLERAVYGANTPIKESLKLERAVYGANTPKESSLDPTQGSHTAENIGKyDLKGPGKKVILELVEQLSLDPTQGSHTAENIGKYDLKGPGKKVILELVEQLLKLERAVYGANTPKES.AP6v1 construct: 3-1-2-1-2-3-3-2-1 (SEQ ID NO: 18)LKLERWLKLERAVYGANTPKESGKYDLKGPGKKVILELEVQLSLDPTQGSHTAENIGKYDLKGPGKKVILELEVQLSLDPTQGSHTAENILKLERAVYGANTPKESLKLERAVYGANTPKESSLDPTQGSHTAENIGKyDLKGPGKKVILELEVQL.AP6v2 construct: 3-1-2-1-2-3-3-2-1 (SEQ ID NO: 19)LKLERWLKLERAVYGANTPKESGIKYDLKGPGKKVILELVEQLSLDPTQGSHTAENIGKYDLKGPGKKVILELVEQLSLDPTQGSHTAENILKLERAVYGANTPKESLKLERAVYGANTPKESSLDPTQGSHTAENIGKyDLKGPGKKVILELVEQL.AP7v1 construct: 1-2-3-1-2-3-1-2-3 (SEQ ID NO: 20)GKYDLWGKYDLKGPGKKVILELEVQLSLDPTQGSHTAENILKLERAVYGANTPKESGKYDLKGPGKKVILELEVQLSLDPTQGSHTAENILKLERAVYGANTPKESGKYDLKGPGKKVILELEVQLSLDPTQGSHTAENILKLERAVYGANTPKES.AP7v2 construct: 1-2-3-1-2-3-1-2-3 (SEQ ID NO: 21)GKYDLWGKYDLKGPGKKVILELVEQLSLDPTQGSHTAENILKLERAVYGANTPKESGKYDLKGPGKKVILELVEQLSLETTQGSHTAENILKLERAVYGANTPKESGKYDLKGPGKKVILELVEQLSLDPTQGSHTAENILKLERAVYGANTPKES. (SEQ ID NO: 22)GKYDLWGKYDLKGPGKKVILELEVQLGKYDLKGPGKKVILELEVQLGKYDLKGPGKKVILELEVQLSLDPTQGSHTAENISLDPTQGSHTAENISLDPTQGSHTAENILKLERAVYGANTPKESLKLERAVYGANTPKESLKLERAVYGANTPKES.AP8v2 construct: 1-1-1-2-2-2-3-3-3 (SEQ ID NO: 23)GKYDLWGKYDLKGPGKKVILELVEQLGKYDLKGPGKKVILELVEQLGKYDLKGPGKKVILELVEQLSLDPTQGSHTAENISLDPTQGSHTAENISLDPTQGSHTAENILKLERAVYGANTPKESLKLERAVYGANTPKESLKLERAVYGANTPKES.

Other Sequences of Interest

Also provided are additional specific Anaplasma (e.g. Aph) proteins andpolypeptides that may be used to elicit or enhance an immune response asdescribed herein. These include the exemplary sequences depicted inFIGS. 4A and B (referred to herein as APH_1235, SEQ ID NO: 25, and P130(APH_0032) SEQ ID NO: 24, as well as variants and antigenic segments orepitopes thereof. P130 (also referred to in the literature as APH_0032,GE130, or AmpB) contributes to Aph virulence and survival in host cells.APH_1235 is expressed by the bacterium exclusively when it is in itsinfectious or dense core (DC) form, and contributes to infectivity.These proteins/polypeptides, and/or subfragments thereof, may be usedalone e.g. in vaccine compositions, as diagnostic tools, etc. asdescribed herein, or one or both of the sequences may be used incombination with one or more chimeritopes. In some aspects, asubfragment of P130 is used, e.g. the exemplary segment spanning aC-terminal portion of the protein from residues 163 to 619, inclusive,of SEQ ID NO: 24. This segment is referred to herein as P130C.

Nucleic Acids and Vectors

Also encompassed by this disclosure are nucleic acid sequences thatencode the amino acid sequences disclosed herein. Such nucleic acidssequences include DNA, RNA, DNA/RNA hybrids, complementary DNA (cDNA),species homologs and variant sequences, and the like. In some aspects,the nucleic acids sequences are DNA.

In some aspects, the nucleic acid sequences presented herein are codonoptimized for a particular production system, e.g. they may be codonoptimized to eliminate rare codons that interfere with production in abacterial expression system. For example, the eight least used codons ofEscherichia coli shown below with the amino acids they encode, can beeliminated:

AGG arginine AGA arginine AUA isoleucine CUA leucine CGA arginine CGGarginine CCC proline UCG

The nucleic acid sequences may comprise or be operably linked to variousnoncoding regulatory elements and/or expression related sequences,examples of which include but are not limited to: stop transfersequences, expression control sequences, expression enhancing sequences,etc. Methods for preparing polynucleotides operably linked to anexpression control sequence and expressing them in a host cell are knownin the art. See, e.g., U.S. Pat. No. 4,366,246, the complete contents ofwhich is hereby incorporated by reference in entirety. A polynucleotideof the disclosure is operably linked when it is positioned adjacent toor close to one or more expression control elements, which directtranscription and/or translation of the polynucleotide.

In addition, the disclosure encompasses vectors which contain or housethe nucleic acid sequences. Examples of suitable vectors include but arenot limited to plasmids, cosmids, viral based vectors, expressionvectors, etc. In some aspects, PCR amplicons are used for production ofthe proteins in a bacterial system.

Production of Proteins

The chimeritopes disclosed herein may be produced by any suitablemethod, many of which are known to those of skill in the art. Forexample, the proteins may be chemically synthesized, or produced usingrecombinant DNA technology i.e. produced by organisms or cells that aregenetically engineered to produce the proteins. Exemplary organisms andcells include but are not limited to bacterial cells; mammalian, yeastand insect cells; plants and plant cells, etc. In addition, productionmay also be via cell-free prokaryotic or eukaryotic-basedtranscription/translation systems, or by other in vitro systems, etc.

Compositions

The disclosure also provides compositions (pharmaceutical compositionssuch as immunogenic compositions, vaccines and compositions for use indiagnostic assays) comprising the chimeritopes disclosed herein and,optionally, one or more additional sequences of interest such as SEQ IDNOS: 25 and 26, for use in eliciting an immune response toAnaplasniataceae species. The compositions generally include one or moretypes of substantially purified chimeritopes as described herein, and apharmacologically suitable carrier. In other words, the chimeritopes inthe composition may all be the same, or may be different so that thecomposition is a “cocktail” of different types of chimeritopes. Thepreparation of such compositions for use as vaccines is well known tothose of skill in the art. Typically, such compositions are preparedeither as liquid solutions or suspensions, however solid forms such astablets, pills, powders and the like are also contemplated. Solid formssuitable for solution in, or suspension in, liquids prior toadministration may also be prepared. The preparation may also beemulsified. The active ingredients may be mixed with excipients whichare pharmaceutically acceptable and compatible with the activeingredients. Suitable excipients are, for example, water, saline,dextrose, glycerol, phosphate buffered saline, Ringer's solution, Hank'ssolution, maltodextrin, ethanol, or the like, singly or in combination,as well as substances such as wetting agents, emulsifying agents,tonicity adjusting agents, detergent, or pH buffering agents. Additionalactive agents, such as bactericidal agents can also be used.Pharmaceutically acceptable salts can also be used in compositions ofthe disclosure, for example, mineral salts such as hydrochlorides,hydrobrom ides, phosphates, or sulfates, as well as salts of organicacids such as acetates, proprionates, malonates, or benzoates.

If it is desired to administer an oral form of the composition, variousthickeners, flavorings, diluents, emulsifiers, dispersing aids orbinders and the like may be added. The composition of the presentdisclosure may contain any such additional ingredients so as to providethe composition in a form suitable for administration. The final amountof chimeric protein in the formulations may vary. However, in general,the amount in the formulations will be from about 0.01-99%,weight/volume.

The vaccine preparations of the present disclosure may further compriseone or more adjuvants, suitable examples of which include but are notlimited to: mineral salts, alum (multiple different substitutedvariants), squalene-based adjuvants (e.g. MF59 adjuvant), muramylpeptide, saponin derivatives, mycobacterium cell wall preparations,certain emulsions, monophosphoryl lipid A, mycolic acid derivatives,nonionic block copolymer surfactants, QUIL A®, cholera toxin B subunit,polyphosphazene and derivatives, immunostimulating complexes (ISCOMs),cytokine adjuvants, lipid adjuvants, mucosal adjuvants, certainbacterial exotoxins and other components, certain oligonucleotides, PLG,SEPPIC™, ALHYDRAGEL®, CpG, cyclic-di-GMP, Freund's adjuvant and others.

Dosage forms of the compositions are also encompassed, especially singledose forms that are suitable for use as a vaccine. The dosage forms maybe injectable, inhalable or oral, depending on the intended route ofadministration.

Antibodies

The disclosure also encompasses antibodies and antigen binding fragmentsthereof that bind to at least one of the recombinant polypeptidesdescribed herein. In particular, the antibodies bind specifically, or atleast selectively, to at least one epitope in the recombinantpolypeptides. For example, an antibody or antigen-binding portionthereof specifically binds to a polypeptide when it exhibits a bindingaffinity K_(a) of 10⁷ l/mol or more. Specific binding can be testedusing, for example, surface plasmon resonance, an enzyme-linkedimmunosorbant assay (ELISA), a radioimmunoassay (RIA), a dot-blot, aslot-blot or a western blot assay using methodology well known in theart.

The antibodies may also bind to a variant polypeptide or a fragment of apolypeptide, so long as the variant or fragment contains at least oneAnaplasma epitope that is or is biologically equivalent to an epitopedisclosed herein. The antibodies may be polyclonal, monoclonal, singlechain antibodies (scFv), or antigen binding fragments thereof, e.g. aportion of an intact antibody comprising the antigen binding site orvariable region of an intact antibody, but free of the constant heavychain domains of the Fc region. Examples include Fab, Fab′, Fab′-SH,F(ab′)₂ and F_(v) fragments. The antibodies may be of any class,including, for example, IgG, IgM, IgA, IgD and IgE and/or any subclass,IgG1, IgG2 etc.

An antibody can be made in vivo in suitable laboratory animals or invitro using recombinant DNA techniques. Means for preparing andcharacterizing antibodies are well known in the art. See, e.g., Dean,Methods Mol. Biol. 80:23-37 (1998); Dean, Methods Mol. Biol. 32:361-79(1994); Baileg, Methods Mol. Biol. 32:381-88 (1994); Gullick, MethodsMol. Biol. 32:389-99 (1994); Drenckhahn et al. Methods Cell. Biol.37:7-56 (1993); Morrison, Ann. Rev. Immunol. 10:239-65 (1992); Wright etal. Crit. Rev. Immunol. 12:125-68 (1992). For example, polyclonalantibodies can be produced by administering a polypeptide of thedisclosure to an animal, such as a human or other primate, mouse, rat,rabbit, guinea pig, goat, pig, dog, cow, sheep, donkey, or horse. Serumfrom the immunized animal is collected and the antibodies are purifiedfrom the plasma. Techniques for producing and processing polyclonalantibodies are known in the art.

In particular, monoclonal antibodies directed against epitopes presentin a polypeptide can be readily produced. For example, normal B cellsfrom a mammal, such as a mouse, which was immunized with a polypeptidecan be fused with, for example, HAT-sensitive mouse myeloma cells toproduce hybridomas. Hybridomas producing specific antibodies can beidentified using radioimmunoassay (RIA) and/or ELISA and isolated bycloning in semi-solid agar or by limiting dilution. Clones producingspecific antibodies are isolated by another round of screening.Monoclonal antibodies can be screened for specificity using standardtechniques, for example, by binding a polypeptide of interest to amicrotiter plate and measuring binding of the monoclonal antibody by anELISA assay. Techniques for producing and processing monoclonalantibodies are known in the art. See e.g., Kohler & Milstein, Nature,256:495 (1975). Particular isotypes of a monoclonal antibody can beprepared directly, by selecting from the initial fusion, or preparedsecondarily, from a parental hybridoma secreting a monoclonal antibodyof a different isotype by using a sib selection technique to isolateclass-switch variants. See Steplewski et al., P.N.A.S. U.S.A. 82:86531985; Spria et al., J. Immunolog. Meth. 74:307, 1984. Monoclonalantibodies of the disclosure can also be recombinant monoclonalantibodies. See, e.g., U.S. Pat. Nos. 4,474,893; 4,816,567. Antibodiescan also be chemically constructed. See, e.g., U.S. Pat. No. 4,676,980.

Accordingly, also encompassed are methods of producing (generating)antibodies to the antigenic sequences disclosed herein. Such methods mayinclude steps of 1) providing or obtaining at least one antigenicchimeritope as disclosed herein; 2) administering the chimeritope to amammal that is capable of generating antibodies to the chimeritope; andafter a period of time sufficient for an antibody-generating immuneresponse to occur within the mammal, 3) harvesting antibodies from themammal.

Antibodies that specifically bind the antigens disclosed herein areparticularly useful for detecting the presence of Anaplasma antigens ina sample, such as a serum, blood, plasma, urine, fecal, tissue, orsaliva sample from a subject, e.g. a mammal. An immunoassay forAnaplasma antigens can utilize one antibody or several differentantibodies. Immunoassay protocols can be based upon, for example,competition, direct reaction, or sandwich type assays using, forexample, labeled antibody. Antibodies of the disclosure can be labeledwith any type of label known in the art, including, for example,fluorescent, chemiluminescent, radioactive, enzyme, colloidal metal,radioisotope and bioluminescent labels. Other antibodies of thedisclosure can specifically bind Aph antigens and Apl (A. platys)antigens, or Aph antigens and other Anaplasma spp. antigens, and can beused as described herein for antibodies that bind to Aph.

Methods Methods of Eliciting an Immune Response

The disclosure also provides methods of eliciting an immune response toAnaplasma by administering a composition comprising one or more types ofthe chimeritope proteins disclosed herein. The composition is generallyadministered in an amount sufficient to elicit an immune response, e.g.a therapeutic dose is administered. An immune response (reaction) is aresponse to an antigen that occurs when lymphocytes identify theantigenic molecule as foreign and induce the formation of antibodies andlymphocytes capable of reacting with it and, in some aspects, renderingit harmless. In this activation process the main cells involved are Tcells and B cells (sub-types of lymphocytes), and macrophages (a type ofleucocyte or white blood cell). These cells produce cytokines thatinfluence the activity of other immune cells. B cells, when activated byhelper T cells undergo clonal expansion and differentiate into effectorB cells, which are short lived and secrete antibodies, and memory Bcells, which are long lived and produce a fast, remembered response whenexposed to the same infection in the future. B cells mature to produceimmunoglobulins (also known as antibodies), that react with (bind to)antigens. At the same time, macrophages process antigens intoimmunogenic units that can stimulate B lymphocytes to differentiate intoantibody-secreting plasma cells, stimulating the T cells to releaselymphokines. Complement is a group of normal serum proteins that enhancethe immune response by becoming activated as the result ofantigen-antibody interaction. The first contact with any antigensensitizes the affected individual and promotes a primary immuneresponse. Subsequent exposure of a sensitized individual to the sameantigen results in a more rapid and massive reaction, called thesecondary immune response (“booster response” or the “anamnesticreaction”). An anamnestic response manifests in the form of increasedlevels of circulating antibody.

Thus, methods of administering the compositions described herein mayinclude e.g. an initial administration, followed by follow-upadministrations at suitable time intervals, e.g. after about 3 to 12weeks, and/or after about 6 months, and also optionally e.g. annually,or every 5 or 10 years thereafter to maintain a high level ofprotection.

The vaccine preparations of the present disclosure, or the nucleotidesthat encode them, may be administered by any of the many suitable meanswhich are well known to those of skill in the art, including but notlimited to: by injection, inhalation, orally, intranasally, intradermalinjection as part of a DNA based vaccine, by ingestion of a food productcontaining the chimeric protein, etc. In general, the mode ofadministration is subcutaneous, intramuscular or oral. In addition, thecompositions may be administered in conjunction with other treatmentmodalities such as substances that boost the immune system,chemotherapeutic agents (e.g. antibiotics), and the like.

The chimeritopes disclosed herein elicit an immune response whenadministered to a subject. Generally, the immune response involves theelements described above, including the elicitation of antibodies. Insome aspects, the immune response is a protective immune response, i.e.after at least one administration of one dose of a vaccine preparationas described herein, and typically after two or more doses areadministered, if the vaccinated individual is exposed to an infectiousagent comprising the antigens present in a chimeritope (e.g. anAnaplasmataceae bacteria), the subject's immune system recognizes anddestroys the infectious agent before an infection is established. Inother aspects, the immune response may not be fully protective, but atleast slows or decreases the level of infection established by thebacterium.

The vaccines are useful to inoculate naïve individuals (those who havenot been exposed to or infected by Anaplasmataceae bacteria) and canalso be beneficial to those who have been exposed and/or who are alreadyinfected. For example, administration of the vaccine may curb thepotential of the bacteria to establish an infection, or may slow orgradually eradicate bacteria already present in the individual, therebylessening one or more symptoms of disease.

Diagnostic Methods

The chimeritopes of the disclosure can be used to detect antibodies orantibody fragments specific for Anaplasma spp. in a test sample, such asa biological sample, an environmental sample, or a laboratory sample,from a subject. A biological sample can include, for example, sera,saliva, blood, cells, plasma, urine, feces, or tissue from a mammal suchas a horse, cat, dog or human. The test sample can be untreated,precipitated, fractionated, separated, diluted, concentrated, orpurified. Subjects who are tested using these methods may beasymptomatic or symptomatic with respect to exhibiting symptoms ofanaplasmosis.

In one aspect, methods of the disclosure comprise contacting one or morerecombinant polypeptides of the disclosure with a test sample underconditions that allow antigen/antibody complexes, i.e., immunecomplexes, to form between the polypeptides and antibodies that arepresent in the sample, and then detecting the complexes. Assays andconditions that are used to detect antibody/polypeptide complexes aregenerally known in the art.

Alternatively, antibodies disclosed herein can be used in a method ofdiagnosing Anaplasma infection in a subject e.g., a human or animalsuspected of having an Anaplasma infection. A suitable test sample isobtained from the subject and the test sample is contacted with one ormore antibodies under conditions enabling the formation ofantibody-antigen complexes between the antibodies and Anaplasma bacteria(or fragments or polypeptides thereof) and then detecting the complexes.Assays and conditions that are used to detect antibody/polypeptidecomplexes are generally known in the art.

The detection of antigen/antibody complexes is an indication that themammal has an Anaplasma infection whereas the absence of immunecomplexes represents a negative result. The amount of antibody/antigencomplex can be determined by methodology known in the art, andcomparisons to positive and negative controls are generally employed,e.g. to establish a frame of reference, to establish as baseline, etc.

In some aspects, the antigen/antibody are detected indirectly when anindicator reagent or detectable label comprising a signal generatingmoiety is detected, e.g. a chromophore or enzyme substrate that isattached directly or indirectly to the polypeptide/antibody complexes.Those of skill in the art are familiar with such detection schemes, e.g.colorimetric labels, second and third anti-species antibodies, the useof enzymes and enzyme substrates, etc. Assays of the disclosure include,but are not limited to those based on competition, direct reaction orsandwich-type assays, including, but not limited to enzyme linkedimmunosorbent assay (ELISA), dot blot, slot blot, western blot, IFA,radioimmunoassay (RIA), hemagglutination (HA), fluorescence polarizationimmunoassay (FPIA), and microtiter plate assays (any assay done in oneor more wells of a microtiter plate).

Assays can use solid phases or substrates or can be performed byimmunoprecipitation or other methods that do not utilize solid phases.Where a solid phase or substrate is used, one or more recombinantpolypeptides or antibodies of the disclosure are directly or indirectlyattached to a solid support or a substrate such as a microtiter well,magnetic bead, non-magnetic bead, bar, matrix, membrane, fibrous matcomposed of synthetic or natural fibers (e.g., glass or cellulose-basedmaterials or thermoplastic polymers, such as, polyethylene,polypropylene, or polyester), sintered structure composed of particulatematerials (e.g., glass or various thermoplastic polymers), or castmembrane film composed of nitrocellulose, nylon, polysulfone or the like(generally synthetic in nature). The substrate materials are used insuitable shapes, such as films, sheets, or plates, or are coated onto orbonded or laminated to appropriate inert carriers, such as paper, glass,plastic films, or fabrics. Suitable methods for immobilizing peptides onsolid phases include ionic, hydrophobic, covalent interactions and thelike.

The formation of a polypeptide/antibody complex or animmunocomplex/indicator complex can be detected by e.g., radiometric,colorimetric, fluorometric, size-separation, or precipitation methods.Optionally, detection of a polypeptide/antibody complex is by theaddition of a secondary antibody that is coupled to an indicator reagentcomprising a signal generating compound. Indicator reagents comprisingsignal generating compounds (labels) associated with apolypeptide/antibody complex can be detected using the methods describedabove and include chromogenic agents, catalysts such as enzymeconjugates fluorescent compounds such as fluorescein and rhodamine,chemiluminescent compounds such as dioxetanes, acridiniums,phenanthridiniums, ruthenium, and luminol, radioactive elements, directvisual labels, as well as cofactors, inhibitors, magnetic particles, andthe like. Examples of enzyme conjugates include alkaline phosphatase,horseradish peroxidase, beta-galactosidase, and the like. The label iscapable of producing a detectable signal either by itself or inconjunction with one or more additional substances.

Formation and detection of antigen/antibody is indicative of thepresence of anti-Anaplasma spp. antibodies in the sample (if therecombinant chimeritopes are used in the assay) or of the presence ofAnaplasma spp. in the sample (if antibodies are used in the assay).Either way, the methods of the disclosure are used to diagnoseanaplasmosis in a subject. The methods of the disclosure can alsoindicate the amount or quantity of anti-Anaplasma spp. antibodies orAnaplasma spp. in a test sample. Generally, the amount of antibodycomplex that is present is proportional to the signal generated.

The disclosure further comprises assay kits (e.g., articles ofmanufacture) for detecting levels of circulating antibody that wereinduced by vaccination, anti-Anaplasma spp. antibodies orantigen-binding antibody fragments in a sample. A kit comprises one ormore chimeritopes of the disclosure and means for determining binding ofthe chimeritopes to anti-Anaplasma spp. antibodies or antigen-bindingantibody fragments in the sample; and/or anti Anaplasma antibodiesgenerated against the chimeritopes disclosed herein. Other componentssuch as buffers, controls, and the like, known to those of ordinaryskill in art, are generally included in such test kits.

In addition, the assays described herein may include reagents thatdetect other pathogens, e.g. heartworm and/or B. burgdorferi, E.chaffeensis, and/or E. canis. Thus, an assay may detect multiplepathogens in a single sample.

It is to be understood that this invention is not limited to particularembodiments described herein above and below, and as such may, ofcourse, vary. It is also to be understood that the terminology usedherein is for the purpose of describing particular embodiments only, andis not intended to be limiting.

Where a range of values is provided, it is understood that eachintervening value between the upper and lower limit of that range (to atenth of the unit of the lower limit) is included in the range andencompassed within the invention, unless the context or descriptionclearly dictates otherwise. In addition, smaller ranges between any twovalues in the range are encompassed, unless the context or descriptionclearly indicates otherwise.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Representative illustrativemethods and materials are herein described; methods and materialssimilar or equivalent to those described herein can also be used in thepractice or testing of the present invention.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference, and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention. Further, the dates ofpublication provided may be different from the actual dates of publicavailability and may need to be independently confirmed.

It is noted that, as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as support for the recitation in the claims of suchexclusive terminology as “solely,” “only” and the like in connectionwith the recitation of claim elements, or use of a “negative”limitations, such as “wherein [a particular feature or element] isabsent”, or “except for [a particular feature or element]”, or “wherein[a particular feature or element] is not present (included, etc.) . . .”.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

EXAMPLES Example 1. Expression and Production of Recombinant AP3v2 andAP4v2

The DNA sequences that encode for AP3v2 and AP4v2 chimeric proteins werecodon-optimized, synthesized, and cloned into expression vectors pET28b(MilliporeSigma; Burlington, Mass.) and pFLEX30 (proprietary to Zoetis)by Blue Heron Biotech (Bothell, Wash.). pFLEX30 utilizes aheat-inducible promotor for expression of the target protein. Eachconstruct encodes for a N-terminal 6×His tag, which allows forpurification of the expressed protein via a Ni²⁺ column. Plasmidconstructs containing sequences encoding for AP3v2 and AP4v2 weretransformed into E. coli expression hosts BL21(DE3)Star (for pET28b) andBL21 (for pFLEX30). Designations for the constructs are as follows:

ZRL309=BL21(DE3)Star/pET28b/AP3v2

ZRL310=BL21(DE3)Star/pET28b/AP4v2

ZRL311=BL21/pFLEX30/AP3v2

ZRL312=BL21/pFLEX30/AP4v2

All initial expression studies were carried out in Terrific Broth(Teknova; Hollister, Calif.) containing 50 ug/ml kanamycin, at the 100ml scale in baffled shake flasks while shaking at 200 RPM. These studieswere followed by larger scale (500 ml) expression in TB using 2 Lbaffled shake flasks. All pET28b constructs were propagated at 37° C. toan ˜OD₆₀₀ 3.0, at which time they were induced with 1 mM IPTG (Time 0;TO). All pFLEX30 constructs were propagated at 33° C. to an ˜OD₆₀₀ 3.0,followed immediately by a 42° C. heat induction at TO. All pFLEX30 andpET28b cultures were allowed to continue growing for an additional 2 or3 hrs post-induction. The cells were then recovered by centrifugation(10 min, 8,500×G) and frozen (−20° C.). As needed the frozen sampleswere thawed, mixed with solubilizing solution and boiled. Samples wereevaluated for production of the recombinant proteins by electrophoresison Novex precast 4-12% SDS PAGE gels (ThermoFisher Scientific; Waltham,Mass.). Protein production over time was monitored. The results forAP3v2 are shown in FIGS. 1A and 1B, and the results for AP4v2 are shownin FIGS. 2A and 2B. pET28b expression of both AP proteins appeared to be“leaky”, as small amounts of AP3v2 and AP4v2 were visible prior to IPTGinduction. The pFLEX30 expression system was therefore used for furthercloning and protein production due to the ability to better controlexpression from this vector.

To purify the chimeritope proteins, frozen cell pellets were resuspendedin 200 mls of 50 mM Tris HCl (pH 8.0). Re-suspended cells were lysed bypassing once through an Avestin C3 cell disruptor (Avestin Inc.; Ottawa,ON, Canada) at 25,000 PSI. Following homogenization, the lysed cellslurry was centrifuged at 10,000×G for 30 min at 4° C. Once spunsupernatant was poured off, the pellet was re-suspended in equilibrationbuffer (50 mM Tris; 10 mM NaCl; 6M Urea; 10 mM imidazole, pH 8.0) andloaded onto a Ni SEPHAROSE™ Excel 5 mL XK16 column (GE Healthcare LifeSciences; Pittsburgh, Pa.), and purified using the ÄKTA™ pure proteinpurification system (GE Healthcare Life Sciences). The column was washedwith equilibration buffer until absorption of UV light was at baseline.Elution was then conducted using a 0-100% B gradient over 5 columnvolumes using an elution buffer (50 mM Tris; 10 mM NaCl; 6M Urea; 500 mMimidazole, pH 8.0). Fractions were collected and select fractions werepooled and dialyzed into 50 mM Tris 10 mM NaCl (pH 8.0). The Ap3v2protein (before and after filtration through a 0.2 urn filter) is shownin FIG. 3. The results demonstrate that the proteins can be readilypurified and that their yield and integrity is not affected bysterilization filtration.

Example 2. Cloning, Expression, Purification and Antigenicity ofAPH_1235 and P130

APH_1235, full-length P130 (P130FL) and a C-terminal antigenic domain ofP130 were PCR amplified from previous cloning vectors and annealed withlinearized pET45 Ligase Independent Cloning vector used standardconditions. The annealed DNA was transformed into E. coli NOVABluecells, and the plasmids propagated. The plasmids were then purified andintroduced into E. coli BL21/DE3 cells. Protein production was inducedusing IPTG. Cell lysates from pre and post-induced cultures werefractionated by SDS-PAGE and the gels stained to visualize the proteins.FIGS. 5A and 5B show the induction results for the APH_1235 and P130FLproteins, respectively (data not shown for P130C). After determiningthat the proteins fractionated into the soluble phase of the celllysates, the proteins were purified using and AKTA purification platformand Ni²⁺ affinity chromatography; they were then analyzed by SDS-PAGEelectrophoresis (FIGS. 5C and 5D). Note that the amino acid sequences ofAPH_1235 and P130 are shown in FIG. 4 for reference.

Recombinant P130C (C-terminal domain), P130FL (full-length protein) orAPH_1235 (full-length protein) were screened by standard single dilutionELISA with serum from healthy or Aph infected dogs. The proteins wereimmobilized in the wells of ELISA plates, non-specific binding wasblocked and the canine serum samples were added at a 1:200 dilution.Antibody binding was detected using horseradish peroxidase conjugatedgoat anti-canine IgG secondary antibody, and chemiluminescence (FIG.6A). These data demonstrate that infected canines develop an IgGresponse to P130 (both full length and C-terminal domains) and APH_1235during natural infection. These results demonstrate that the P130 andAPH_1235 proteins are antigenic during infection in canines.

While the AP constructs are designed proteins (not natural proteins), ifthe epitopes that comprise the chimeritopes are presented on the Aphcell surface by OmpA, Asp14 and AipA, then these epitopes should triggeran antibody response during infection and that antibody should be ableto bind to the AP proteins. To test this, recombinant AP3v1 and AP4v1were immobilized in ELISA plate wells and screened with serum from Aphinfected dogs. Note that recombinant P44 protein served as a positivecontrol for antibody binding. P44 has been demonstrated to consistentlyinduce antibody formation in infected mammals. P44 and the AP proteinswere bound by antibody present in serum of infected dogs (FIG. 6B).These analyses revealed several important findings. First, the epitopesthat were selected for inclusion in the AP constructs are naturallyantigenic and are presented on the cell surface. Second, when theepitopes are isolated from their proteins of origin and presented in thecontext of chimeritopes, they retain the ability to bind to antibodythat develops during natural infection. Lastly, from this it can beconcluded that antibody elicited by vaccination with the AP chimeritopeswill bind to the epitopes of OmpA, Asp14 and AipA as presented on thecell surface of Aph.

Example 3. Generation of Antiserum Against the AP Chimeritopes in BeagleDogs Using Novel Vaccine Formulations

The objective of this study was to generate immune serum in dogs againstthe AP chimeritopes and determine if the canine hyperimmune sera canblock Aph invasion of HL60 cells at levels similar to that observed withthe analogous anti-AP antisera generated in rats and rabbits.Chimeritopes (AP1 v1 to AP4v1) were formulated in REHYDRAGEL® orQUILA®/Cholesterol/CpG for inoculation of dogs. Twenty-seven femalepurpose-bred Beagles, ˜21 weeks of age at day 0, and seronegative for E.canis and A. phagocytophilum, were randomly assigned to nine treatmentgroups (T01-T09; 3 dogs/treatment group). All dogs were vaccinatedaccording to the study design presented in the table below. Blood wascollected as indicated with terminal blood samples collected on days 54or 55.

STUDY DESIGN Treat- ment # of Group Dogs Treatment Day Dose Route T01 3AP1v1 + REHYDRAGEL ® 0, 1 ml SC T02 3 AP2v1 + REHYDRAGEL ® 21, 1 ml SCT03 3 AP3v1 + REHYDRAGEL ® 42 1 ml SC T04 3 AP4v1 + REHYDRAGEL ® 1 ml SCT05 3 AP1v1 + QUIL A ® + 1 ml SC Cholesterol/CpG T06 3 AP2v1 + QUILA ® + 1 ml SC Cholesterol/CpG T07 3 AP3v1 + QUIL A ® + 1 ml SCCholesterol/CpG T08 3 AP4v1 + QUIL A ® + 1 ml SC Cholesterol/CpG T09 3AP1v1 + AP2v1 + AP3v1 + 1 ml SC AP4v1 + QUIL A ® + Cholesterol/CpGEach AP chimeritope was formulated at 50 μg/dose in a total volume of 1ml. The study was conducted according to the protocol with the followingexceptions: 1) Injection site observations on day 42 (pre-vaccination)through day 49 (left and right neck) were added. 2) Additional weeklyobservations were added for reactions at the injection site thatcontinued beyond the seven-day observation period. 3) Half of the dogswere terminated on day 54, and the remaining on day 55.

The following were deviations from the protocol: 1) blood was to becollected on day 0, 21, 35, 42 and 56 of study (2 on days 0, 21, 35, 42and final large bleed 200 mLs on day 56/dog). 2) Blood was collected onday 36 (not day 35). 3) Body temperatures were determined pre- andpost-vaccination on day 0 through 7, 21-28 and 42-49. Body temperatureswere also determined at the time an animal was removed from study (day55 or 56). No adverse events occurred during the duration of this study.

The results of this study demonstrated that AP1, AP2, AP3v1 and AP4v1induced high IgG titer antibody responses in canines as measured byendpoint dilution ELISA (data not shown). Some dogs were found to havelow level titers to some AP proteins prior to vaccination. The originsof this background binding are not clear. Serum samples with highbackground levels of antibody were excluded from further analysis. Asdescribed below, the serum samples were then pooled and used in blockingexperiments. In these experiments microscopy was employed to assessinfection and the number of Aph vacuoles (ApVs) that form in eachinfected cell.

Example 4. Assessment of the Ability of Anti-Chimeritope Antisera Aloneor in Combination with Anti-APH_1235 and/or P130 Antisera to Inhibit AphInfection of HL60 Cells

To conduct these assays detailed within, cultures of infection free andAph infected HL60 cells are required. The infected and uninfected HL60cells were cultivated in Iscove's modified Dulbecco media (10% FBS; 37C; humified chamber; 5% CO₂). To obtain purified Aph cells, infectedHL60 cells were sonicated and the bacteria were recovered bydifferential centrifugation. To conduct the antibody blockingexperiments, purified Aph cells were incubated with 2.5×10⁶ uninfectedHL-60 cells in the presence of the desired hyperimmune serum derivedanimals (rat, rabbit or dogs) vaccinated with individual AP proteins,combinations of AP protein, P130 and APH_1235. The antisera were used atdilutions ranging from 1:5 to 1:625 (as indicated in each experiment).Negative controls consisted of preimmune serum or anti-OspC antiserum.After combining the cells and desired sera, the samples were gentlymixed. After the incubation period, the unbound bacteria were removed bywashing and the population of infected and uninfected HL60 cells wererecovered by centrifugation. The cells were suspended at 250,000 cellsper ml and incubated as above. At 24 h, aliquots of 35,000 cells wereplaced on slides, fixed and permeabilized using ice cold methanol. Toperform IFA analyses, the slides were incubated with 5% bovine serumalbumin (BSA) in PBS for 1 h; washed, and incubated with rabbit anti-P44antiserum (1:500 dilution; PBS with 1% BSA; 30 min). The slides werethen washed with PBS, incubated with Alexa Fluor-488 conjugated goatanti-rabbit IgG (in PBS with 1% BSA; 30 min), washed, and mounted withPROLONG® Gold Antifade medium containing 4′,6-diamidino-2-phenylindole(DAP1). The percentage of cells with at least one ApV was determined byanalysis of 100 cells in triplicate. Similarly, the number of ApVs percell was also determined. To test for significant differences amonggroups, one-way analysis of variance was determined using Tukey's posthoc test (Prism 5.0; GraphPad; San Diego, Calif.) and to assessstatistical significance among pairs the student's t-test was employed(P values of <0.05 were set).

RESULTS: Rat anti-AP1v1, APv2, AP3v1 and APv2 antisera significantlyreduced the percentage of infected cells (FIG. 7A) and the mean numberof ApVs per cell (FIG. 7B) in a dose-dependent manner. Antisera dilutionranging from 1:5 to 1:125 were tested. The negative control sera(preimmune and anti-OspC antisera) had no effect. Antibody to AP3v1 andAP4v1 displayed the most efficient blocking (see FIGS. 7A and 7B at the1:5 dilution). Thus, immunization of rats against chimeritopes bearingthe binding domain sequences of OmpA, Asp14, and AipA promotesproduction of antibodies that can significantly reduce the ability ofAph to infect host cells.

It was next examined if immunization of dogs with AP1v1, AP2v1, AP3v1,AP4v1, or a combination of all four chimeritopes, elicits antibodiesthat interfere with Aph infectivity (FIG. 8A) or mean numbers of ApVsper cell (FIG. 8B). Antisera were generated using two differentadjuvants: REHYDRAGEL® adjuvant or QUILA®/cholesterol/CpG (QCT). Aftergenerating the antisera, Aph bacteria were incubated with HL-60 cellsfor 1 h in the presence of 1:5 dilutions of pooled sera obtained fromdogs that had been immunized with each individual chimeritope, or acombination of all four. Preimmune canine serum served as a negativecontrol. Examination at 24 h post-infection revealed that canineantisera against AP1v1, AP2v1, AP3v1, AP4v1, or all four APv1chimeritopes significantly reduced the percentage of infected cells(FIG. 8A) and lowered the number of ApVs per cell (FIG. 8B). The mosteffective inhibitory activity was observed with antisera elicited byAP3v1, AP4v1, or all four chimeritopes in combination. These datademonstrate that immunization of dogs with the AP constructs inducesproduction of antibodies capable of inhibiting Aph infection of hostcells.

To determine if antisera raised against additional virulence factors ofAph, specifically P130 and APH_1235 could enhance the infection blockingactivity of the anti-AP antisera we turned back to the rat model forinitial analyses. To test this, we focused on rat anti-AP4v1 antisera.Aph bacteria were incubated with HL-60 cells in the presence of 1:5dilutions of rat anti-AP4v1 antiserum, rabbit antiP130 antiserum, rabbitanti-APH_1235 antiserum, antisera against AP4v1 and P130, AP4v1 andAPH_1235, all three antisera, or preimmune serum (see FIG. 9A and FIG.9B). The number of infected cells and the mean number of ApVs per 100cells were determined after 24 and 72 h. The most effectiveinfection-blocking activity was observed for theanti-AP4v1-P130-APH_1235 antisera combination. Therefore, inclusion ofantisera specific for APH_1235 and/or P130 augments and enhances theinhibitory activity of AP4v1 antisera against Aph infection of hostcells.

In a manner similar to the experiments detailed above that used antiseragenerated in rats, we next determined if the blocking ability of canineanti-AP4v1 antiserum could also be improved by combining it withanti-P130 and/or anti-APH_1235 antisera (FIG. 10A-D). Note that theanti-APH_1235 and anti-P130 antisera used in this experiment weregenerated in rabbits. Aph bacteria were incubated with HL-60 cells inthe presence of canine anti-AP4v1 antiserum alone, rabbit anti-P130antiserum alone, rabbit anti-APH_1235 antiserum alone, antisera againstAP4v1 and P130, AP4v1 and APH_1235, or all three antisera. The mosteffective infection blocking capability was observed for theanti-AP4v1-P130-APH_1235 antisera combination. Overall these dataconfirm that canine anti-AP4v1 antiserum's inhibitory capability againstAph infection is augmented by the addition of anti-APH_1235 and/oranti-P130 antisera.

AP1v1, AP2v1, AP3v1, and AP4v1 consist of arrangements of epitopes ofthe following sequences: Asp14 (LKLERAVYGANTPKES; SEQ ID NO: 4); AipA(SLDPTQGSHTAENI; SEQ ID NO: 3) and OmpA (GKYDLKGPGKKVILELEVQL; SEQ IDNO: 1). In the context of the AP3 and AP4 chimeritopes, a second variantof the OmpA epitope sequence was tested (GKYDLKGPGKKVILELVEQL; SEQ IDNO: 2). The difference between SEQ ID NO:1 and 2 is subtle and consistsof a reversal of the two amino acids that are underlined. To determineif this sequence difference might impact blocking ability of the anti-APantisera, rats were immunized with AP3v2 and AP4v2. The anti-AP3v2 andanti-AP4v2 effectively inhibited Aph infection in a dose-dependentmanner at levels similar to that of the anti-AP3v1 and anti-AP4v1 (FIG.11).

As detailed above, when antisera from rats vaccinated with AP4v1 wascombined with anti-P130 and anti-APH_1235 antisera, optimal infectionblocking activity was observed. To be complete, we sought to determineif this would also be the case if rat anti-AP4v2 was combined withanti-P130, anti-APH_1235 antisera or combinations thereof. The mosteffective infection blocking capability was observed for theanti-AP4v2-P130-APH_1235 antisera combination. Note that as anadditional control set in this experiment, in some samples theanti-APH_1235 and anti-P130 antisera was replaced with anti-P44antisera. The purpose of swapping anti-P44 antiserum for anti-APH_1235or P130 antiserum was to determine if the synergistic blocking observedwhen anti-AP4v2 antiserum was combined with anti-APH_1235 and anti-P130antisera was due to antibody specific mediated inhibition resultedsimply from the coating of bacteria with antibodies that stericallyhinder bacterial access to the host cell surface. Theanti-AP4v2-P130-APH_1235 antisera cocktail proved to be significantlymore effective at reducing Aph infection and vacuole numbers thanantiserum cocktails containing anti-P44 antibody (FIG. 12).Collectively, the data presented above demonstrate that a multi-target,multi-valent approach is required to efficiently inhibit the ability ofAph to invade host cells and establish a productive infection.

Example 5. Evaluation of AP Vaccine Formulations in Beagle Dogs

The objective of this study is to evaluate two different vaccineformulations in dogs. One formulation consists of two different APchimeritopes (AP3v2 and AP4v2) and the other of the same twochimeritopes in combination with APH_1235 and P130. Both vaccineformulations are adjuvanted with QUIL A®/Cholesterol/CpG.

Thirty-six (36) female purpose-bred Beagles, approximately 21 weeks ofage at Day 0, and seronegative for E. canis and Aph, are randomlyassigned to three treatment groups (T01-T03, 12 dogs/treatment group).All dogs are vaccinated according to the study design on Days 0 and 21.Blood is collected on Days 0, 21, and 42 (prior to vaccinations), andthen every 3 days starting at Day 45 through the end of the study.

Study Design

# Vaccination Group dogs Treatment Days Dose Adjuvant Route ChallengeBlood Collection T01 12 Placebo 0, 21 1.0 ml — SQ Day 42 Day 0, 21, T0212 AP3v2 + AP4v2 1.0 ml QCT* 42, every 3 T03 12 AP3v2 + AP4v2 + 1.0 mlQCT  days starting APH_1235 + P130 on Day 45 through the end of thestudy *QCT = QuilA ®/Cholesterol/CpG

The primary variable assessed for this study is the presence and theduration of thrombocytopenia post-challenge. Clinical signsmonitored/measured include at least fever, lethargy, depression, swollenlymph nodes, and bleeding. The administration of two chimeritopes(AP3v2; AP4v2), and the same two chimeritopes in combination with twoother Aph antigens (APH_1235; P130) causes a reduction in at least onemeasured post challenge clinical variable.

Example 6. Generation of Monoclonal Antibodies Recognizing AP4v2

Four Balb/c mice are immunized with AP4v2 protein at Maine BiotechnologyServices (Portland, Me.) according to their optimized, proprietary MBSRapid Immunization Multiple Sites (RIMMS) protocol. Mice are immunized 4or 5 times within a 20-day period, at multiple sites on each animal, oneach injection date. The primary immunization is prepared 1:1 inFreund's Complete Adjuvant (FCA); subsequent boosts are prepared inFreund's Incomplete Adjuvant (IFA). At day 20, test bleeds are taken andscreened by ELISA, to determine the antibody titer. If the titers fromany of the mice are sufficiently high, that mouse is selected forfusion. If all the mouse titers are insufficient for fusion, the micereceive an additional boost of antigen (100 μg in IFA). Ten daysfollowing the boost, a test bleed is taken and screened by ELISA. Thiscycle is repeated, if necessary, until at least one mouse is at asufficient titer warranting fusion. The selected mouse receives anunadjuvanted protein boost (resuspended in saline) four days prior tofusion. Splenocytes removed from the mouse are fused with SP2/0 mousemyeloma cells. Following the fusion, the resulting products aredistributed among twenty 96-well plates, and allowed to grow intocolonies. Following this, the 96-well plates are doubled-screened inELISA assays with both a positive screen (AP4v2 protein) and a negativescreen (irrelevant 6×His-tagged protein). Colonies that are positive forthe AP4v2 protein, but negative for the irrelevant 6×His-tagged protein,are selected for scale-up.

While the invention has been described in terms of its several exemplaryembodiments, those skilled in the art will recognize that the inventioncan be practiced with modification within the spirit and scope of theappended claims. Accordingly, the present invention should not belimited to the embodiments as described above, but should furtherinclude all modifications and equivalents thereof within the spirit andscope of the description provided herein.

We claim:
 1. A recombinant, chimeric polypeptide comprising, at leastone copy of an invasion domain/epitope of Anaplasma OmpA, at least onecopy of an invasion domain/epitope of Anaplasma AipA, and at least onecopy of an invasion domain/epitope of Anaplasma Asp14.
 2. Therecombinant, chimeric polypeptide of claim 1, wherein the invasiondomain/epitope of Anaplasma OmpA has a sequence GKYDLKGPGKKVILELEVQL(SEQ ID NO: 1) or GKYDLKGPGKKVILELVEQL (SEQ ID NO: 2).
 3. Therecombinant, chimeric polypeptide of claim 1, wherein the invasiondomain/epitope of Anaplasma AipA has a sequence SLDPTQGSHTAENI (SEQ IDNO: 3).
 4. The recombinant, chimeric polypeptide of claim 1, wherein theinvasion domain/epitope of Anaplasma Asp14 has a sequenceLKLERAVYGANTPKES (SEQ ID NO: 4).
 5. The recombinant, chimericpolypeptide of claim 1, wherein the recombinant, chimeric polypeptidefurther comprises at least one cap sequence.
 6. The recombinant,chimeric polypeptide of claim 1, wherein the at least one cap sequencehas an amino acid sequence that is at least 33% proline and has a randomcoil configuration.
 7. The recombinant, chimeric polypeptide of claim 5,wherein the cap sequence is an OspC sequence PVVAESPKKP (SEQ ID NO: 5).8. The recombinant, chimeric polypeptide of claim 1, wherein the aminoacid sequence of the recombinant, chimeric polypeptide is selected fromthe group consisting of: (SEQ ID NO: 8)GKYDLWGKYDLKGPGKKVILELEVQLSLDPTQGSHTAENILKLERAVYGANTPKESLKLERAVYGANTPKESSLDPTQGSHTAENIGKYDLKGPGKKVILELEVQLSLDPTQGSHTAENIGKYDLKGPGKKVILELEVQLLKLERAVYGANTPKESPVVAESPKKP; (SEQ ID NO: 9)GKYDLWGKYDLKGPGKKVILELVEQLSLDPTQGSHTAENILKLERAVYGANTPKESLKLERAVYGANTPKESSLDPTQGSHTAENIGKYDLKGPGKKVILELVEQLSLDPTQGSHTAENIGKYDLKGPGKKVILELVEQLLKLERAVYGANTPKESPVVAESPKKP; (SEQ ID NO: 10)LKLERWLKLERAVYGANTPKESGKYDLKGPGKKVILELEVQLSLDPTQGSHTAENIGKYDLKGPGKKVILELEVQLSLDPTQGSHTAENILKLERAVYGANTPKESLKLERAVYGANTPKESSLDPTQGSHTAENIGKYDLKGPGKKVILELEVQLPVVAESPKKP; (SEQ ID NO: 11)LKLERWLKLERAVYGANTPKESGKYDLKGPGKKVILELVEQLSLDPTQGSHTAENIGKYDLKGPGKKVILELVEQLSLDPTQGSHTAENILKLERAVYGANTPKESLKLERAVYGANTPKESSLDPTQGSHTAENIGKYDLKGPGKKVILELVEQLPVVAESPKKP; (SEQ ID NO: 12)GKYDLWGKYDLKGPGKKVILELEVQLSLDPTQGSHTAENILKLERAVYGANTPKESGKYDLKGPGKKVILELEVQLSLDPTQGSHTAENILKLERAVYGANTPKESGKYDLKGPGKKVILELEVQLSLDPTQGSHTAENILKLERAVYGANTPKESPVVAESPKKP; (SEQ ID NO: 13)GKYDLWGKYDLKGPGKKVILELVEQLSLDPTQGSHTAENILKLERAVYGANTPKESGKYDLKGPGKKVILELVEQLSLDPTQGSHTAENILKLERAVYGANTPKESGKYDLKGPGKKVILELVEQLSLDPTQGSHTAENILKLERAVYGANTPKESPVVAESPKKP; (SEQ ID NO: 14)GKYDLWGKYDLKGPGKKVILELEVQLGKYDLKGPGKKVILELEVQLGKYDLKGPGKKVILELEVQLSLDPTQGSHTAENISLDPTQGSHTAENISLDPTQGSHTAENILKLERAVYGANTPKESLKLERAVYGANTPKESLKLERAVYGANTPKESPVVAESPKKP; (SEQ ID NO: 15)GKYDLWGKYDLKGPGKKVILELVEQLGKYDLKGPGKKVILELVEQLGKYDLKGPGKKVILELVEQLSLDPTQGSHTAENISLDPTQGSHTAENISLDPTQGSHTAENILKLERAVYGANTPKESLKLERAVYGANTPKESLKLERAVYGANTPKESPVVAESPKKP; (SEQ ID NO: 16)GKYDLWGKYDLKGPGKKVILELEVQLSLDPTQGSHTAENILKLERAVYGANTPKESLKLERAVYGANTPKESSLDPTQGSHTAENIGKYDLKGPGKKVILELEVQLSLDPTQGSHTAENIGKYDLKGPGKKVILELEVQLLKLERAVYGANTPKES; (SEQ ID NO: 17)GKYDLWGKYDLKGPGKKVILELVEQLSLDPTQGSHTAENILKLERAVYGANTPKESLKLERAVYGANTPKESSLDPTQGSHTAENIGKYDLKGPGKKVILELVEQLSLDPTQGSHTAENIGKYDLKGPGKKVILELVEQLLKLERAVYGANTPKES; (SEQ ID NO: 18)LKLERWLKLERAVYGANTPKESGKYDLKGPGKKVILELEVQLSLDPTQGSHTAENIGKYDLKGPGKKVILELEVQLSLDPTQGSHTAENILKLERAVYGANTPKESLKLERAVYGANTPKESSLDPTQGSHTAENIGKYDLKGPGKKVILELEVQL; (SEQ ID NO: 19)LKLERWLKLERAVYGANTPKESGKYDLKGPGKKVILELVEQLSLDPTQGSHTAENIGKYDLKGPGKKVILELVEQLSLDPTQGSHTAENILKLERAVYGANTPKESLKLERAVYGANTPKESSLDPTQGSHTAENIGKYDLKGPGKKVILELVEQL; (SEQ ID NO: 20)GKYDLWGKYDLKGPGKKVILELEVQLSLDPTQGSHTAENILKLERAVYGANTPKESGKYDLKGPGKKVILELEVQLSLDPTQGSHTAENILKLERAVYGANTPKESGKYDLKGPGKKVILELEVQLSLDPTQGSHTAENILKLERAVYGANTPKES; (SEQ ID NO: 21)GKYDLWGKYDLKGPGKKVILELVEQLSLDPTQGSHTAENILKLERAVYGANTPKESGKYDLKGPGKKVILELVEQLSLDPTQGSHTAENILKLERAVYGANTPKESGKYDLKGPGKKVILELVEQLSLDPTQGSHTAENILKLERAVYGANTPKES; (SEQ ID NO: 22)GKYDLWGKYDLKGPGKKVILELEVQLGKYDLKGPGKKVILELEVQLGKYDLKGPGKKVILELEVQLSLDPTQGSHTAENISLDPTQGSHTAENISLDPTQGSHTAENILKLERAVYGANTPKESLKLERAVYGANTPKESLKLERAVYGANTPKES; and (SEQ ID NO: 23)GKYDLWGKYDLKGPGKKVILELVEQLGKYDLKGPGKKVILELVEQLGKYDLKGPGKKVILELVEQLSLDPTQGSHTAENISLDPTQGSHTAENISLDPTQGSHTAENILKLERAVYGANTPKESLKLERAVYGANTPKESLKLERAVYGANTPKES.


9. The recombinant, chimeric polypeptide of claim 8, wherein the aminoacid sequence of the recombinant, chimeric polypeptide is(SEQ ID NO: 13) GKYDLWGKYDLKGPGKKVILELVEQLSLDPTQGSHTAENILKLERAVYGANTPKESGKYDLKGPGKKVILELVEQLSLDPTQGSHTAENILKLERAVYGANTPKESGKYDLKGPGKKVILELVEQLSLDPTQGSHTAENILKLERAVYGA NTPKESPVVAESPKKP.

or (SEQ ID NO: 15) GKYDLWGKYDLKGPGKKVILELVEQLGKYDLKGPGKKVILELVEQLGKYDLKGPGKKVILELVEQLSLDPTQGSHTAENISLDPTQGSHTAENISLDPTQGSHTAENILKLERAVYGANTPKESLKLERAVYGANTPKESLKLERAVYGA NTPKESPVVAESPKKP.


10. A pharmaceutical composition comprising at least one recombinant,chimeric polypeptide of claim
 1. 11. The pharmaceutical composition ofclaim 10, further comprising one or both of: (SEQ ID NO: 25)MKGKSDSEIR TSSSIRTSSS DDSRSSDDST RIRASKTHPQAPSDNSSILS SEDIESVMRC LEEEYGQKLS SELKKSMREEISTAVPELTR ALIPLLASAS DSDSSSRKLQ EEWVKTFMAI MLPHMQKIVA STQG

and (SEQ ID NO: 24) MFERNIPDTY TGTTAEGSPG LAGGDFSLSS IDFTRDFTIESHRGSSADDP GYISFRDQDG NVMSRFLDVY VANFSLRCKHSPYNNDRMET AAFSLTPDII EPSALLQESH STQNNVEEAVQVTALECPPC NPVPAEEVAP QPSFLSRIIQ AFLWLFTPSSTTDTAEDSKC NSSDTSKCTS ASSESLEQQQ ESVEVQPSVLMSTAPIATEP QNAVVNQVNT TAVQVESSII VPESQHTDVTVLEDTTETIT VDGEYGHFSD IASGEHNNDL PAMLLDEADFTMLLANEESK TLESMPSDSL EDNVQELGTL PLQEGETVSEGNTRESLPTD VSQDSVGVST DLEAHSQEVE TVSEVSTQDSLSTNISQDSV GVSTDLEAHS KGVEIVSEGG TQDSLSADFPINTVESESTD LEAHSQEVET VSEFTQDSLS TNISQDSVGVSTDLEVHSQE VEIVSEGGTQ DSLSTNISQD SVGVSTDLEAHSQEVETVSE FTQDSLSTNI SQDSVGVSTD LEVHSQEVEIVSEGGTQDSL STNISQDSVG VSTDLEAHSK GVEIVSEGGTQDSLSADFPI NTVESESTDL EAHSPEGEIV SEVSTQDAPS TGVEIRFMDR DSDDDVLAL, ora subfragment of SEQ ID NO:
 24.


12. The pharmaceutical composition of claim 11, wherein the subfragmentof SEQ ID NO: 24 is or includes residues 163 to
 619. 13. A method ofeliciting an immune response to Anaplasma in a subject in need thereof,comprising administering to the subject an amount of the pharmaceuticalcomposition of claim 10 sufficient to elicit an immune response in thesubject.
 14. The method of claim 13, wherein the immune response is aprotective immune response.
 15. A method of blocking or attenuating thebinding of Anaplasma to mammalian cells in a subject in need thereof,comprising administering to the subject the pharmaceutical compositionof claim 10, wherein the pharmaceutical composition is administered inan amount sufficient to elicit the production of antibodies that blockor attenuate the binding of Anaplasma to mammalian cells in the subject.